Florigen-activating complex

ABSTRACT

Provided is a crystal of a florigen activation complex, including a florigen, a 14-3-3 protein, and a bZIP transcription factor bound to each other. In addition, flowering of a plant is regulated by controlling mechanisms of interactions among those proteins utilizing the crystal. These are achieved as follows. With attention focused on the fact that Hd3a is bound to FD1 via GF14c to form a florigen activation complex, a crystal of the florigen activation complex is produced, conformational information is obtained through the use of the crystal of the florigen activation complex, and the flowering of a plant is regulated by controlling mechanisms of interactions among the florigen and the like utilizing such conformational information.

TECHNICAL FIELD

The present invention relates to a crystal of a florigen activation complex, including a florigen bound to a bZIP transcription factor via a 14-3-3 protein, and to uses of conformational information on the florigen activation complex obtained from the crystal, and a function of the florigen activation complex.

The present application claims priority from Japanese Patent Application No. 2010-061562, which is incorporated herein by reference.

BACKGROUND ART

Regulation of flowering of plants under various environments is considered to be effective for realizing stable food production and establishment of a recycling-oriented economic society system utilizing plant biomass and the like. A molecular basis for signal transduction of a plant environment response needs to be elucidated in order to establish a technology for regulating flowering of plants.

It was proposed 70 years ago that a florigen was present as an inducer of flowering of plants. In recent years, it has been clarified that the molecular nature for the florigen is a product of an FT gene universally present in higher plants. The florigen is a molecule which is synthesized in leaves when day length becomes optimum for flower-bud formation, moves to the shoot apex, and initiates flowering. In rice, Hd3a corresponds to the florigen and is considered to promote heading, and there has been proposed use of a rice Hd3a gene for regulating flowering of plants (Patent Literature 1 and Non Patent Literature 1).

There is a report that rice Hd3a interacts with a rice 14-3-3 protein GF14c, thereby suppressively controlling flowering (Non Patent Literature 2). In Non Patent Literature 2, it has been found that the rice 14-3-3 protein GF14c interacts with Hd3a, as a result of screening of a protein which interacts with Hd3a by a yeast two-hybrid method. It has been suggested that GF14c is mainly localized in the cytoplasm and a complex of Hd3a and GF14c is mainly present in the cytoplasm. GF14c has been considered to act as a negative regulator of flowering based on, for example, the fact that overexpression of GF14c leads to a delay in flowering.

In Arabidopsis, it is considered that an FT protein as the molecular nature for the florigen interacts with a bZIP transcription factor FD and the like in the nucleus, contributing to expression of floral meristem identity genes (Non Patent Literatures 3 and 4). There is a report on use of a bZIP transcription factor for controlling flowering (Patent Literature 2).

Although there are many research reports on the florigen, an action mechanism of the florigen in floral induction has not been clarified yet.

CITATION LIST Patent Literature

-   [PTL 1] JP 2002-153283 A -   [PTL 2] JP 2003-274972 A

Non Patent Literature

-   [NPL 1] Tamaki S et al. Science (2007) vol. 316 (5827) pp. 1033-6 -   [NPL 2] Purwestri Y A et al. Plant and Cell Physiology 2009 50(3):     429-438 -   [NPL 3] Wigge et al. Science (2005) vol. 309 (5737) pp. 1056-9 -   [NPL 4] Abe et al. Science (2005) vol. 309 (5737) pp. 1052-6

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a crystal of a florigen activation complex, including a florigen bound to a bZIP transcription factor via a 14-3-3 protein. Another object of the present invention is to regulate flowering of a plant by clear lying mechanisms of interactions among those proteins utilizing the crystal and controlling such interactions.

Solution to Problem

The inventors of the present invention have made extensive studies in order to achieve the objects. As a result, the inventors have succeeded in crystallizing a florigen activation complex, including a florigen bound to a bZIP transcription factor via a 14-3-3 protein, and have found that the flowering of a plant can be regulated by controlling an interaction mechanism utilizing conformational information obtained from the crystal of the florigen activation complex. Thus, the present invention has been completed.

That is, the present invention includes the following items.

1. A method of regulating flowering of a plant, the method including promoting and/or suppressing formation of a florigen activation complex including a complex of a florigen, a 14-3-3 protein, and a bZIP transcription factor by affecting a binding site between the florigen and the 14-3-3 protein and/or a binding site between the 14-3-3 protein and the bZIP transcription factor in the florigen activation complex,

in which:

the binding site between the florigen and the 14-3-3 protein includes at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and

the binding site between the 14-3-3 protein and the bZIP transcription factor includes at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO: 3.

2. A method of regulating flowering of a plant according to the above-mentioned item 1, in which the promoting and/or suppressing of the formation of the florigen activation complex includes generating a transformant including at least one of the following proteins (A) to (C): (A) a florigen having a mutation in at least one binding site between the florigen and a 14-3-3 protein; (B) a 14-3-3 protein having a mutation in at least one binding site between a florigen and the 14-3-3 protein and/or at least one binding site between the 14-3-3 protein and a bZIP transcription factor; and (C) a bZIP transcription factor having a mutation in at least one binding site between a 14-3-3 protein and the bZIP transcription factor. 3. A transformant, including at least one of the following proteins (A) to (C): (A) a florigen having a mutation in at least one binding site between the florigen and a 14-3-3 protein; (B) a 14-3-3 protein having a mutation in at least one binding site between a florigen and the 14-3-3 protein and/or at least one binding site between the 14-3-3 protein and a bZIP transcription factor; and (C) a bZIP transcription factor having a mutation in at least one binding site between a 14-3-3 protein and the bZIP transcription factor,

in which:

the binding site between the florigen and the 14-3-3 protein includes at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and

the binding site between the 14-3-3 protein and the bZIP transcription factor includes at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO: 3.

4. A polynucleotide, which encodes at least one of the following proteins (A) to (C): (A) a florigen having a mutation in at least one binding site between the florigen and a 14-3-3 protein; (B) a 14-3-3 protein having a mutation in at least one binding site between a florigen and the 14-3-3 protein and/or at least one binding site between the 14-3-3 protein and a bZIP transcription factor; and (C) a bZIP transcription factor having a mutation in at least one binding site between a 14-3-3 protein and the bZIP transcription factor,

in which:

the binding site between the florigen and the 14-3-3 protein includes at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and

the binding site between the 14-3-3 protein and the bZIP transcription factor includes at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO: 3.

5. A recombinant vector, including at least one of the polynucleotides of the above-mentioned item 4. 6. A method of screening a substance that regulates flowering of a plant, the method including any one of the following steps: (1) a step including bringing a candidate substance into contact with any one of a florigen and a 14-3-3 protein, and bringing the candidate substance into contact with any one of the 14-3-3 protein and the florigen, respectively; and (2) a step including bringing a candidate substance into contact with any one of a 14-3-3 protein to which a florigen is bound or unbound and a bZIP transcription factor, and bringing the candidate substance into contact with any one of the bZIP transcription factor and the 14-3-3 protein to which a florigen is bound or unbound, respectively. 7. A method of screening a substance that regulates flowering of a plant according to the above-mentioned item 6, the method further including the following step of:

selecting a candidate substance that promotes and/or inhibits binding in a binding site between the florigen and the 14-3-3 protein and/or a binding site between the 14-3-3 protein and the bZIP transcription factor in the presence of the candidate substance,

in which:

the binding site between the florigen and the 14-3-3 protein includes at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and

the binding site between the 14-3-3 protein and the bZIP transcription factor includes at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO: 3.

8. A method of screening a substance that regulates flowering of a plant according to the above-mentioned item 6 or 7,

in which:

the florigen includes a florigen polypeptide fragment including an amino acid sequence which includes at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177, in an amino acid sequence set forth in SEQ ID NO: 1;

the 14-3-3 protein includes a 14-3-3 protein polypeptide fragment including an amino acid sequence which includes at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256, in an amino acid sequence set forth in SEQ ID NO: 2; and

the bZIP transcription factor includes a bZIP transcription factor polypeptide fragment including an amino acid sequence which includes at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195, in an amino acid sequence set forth in SEQ ID NO: 3.

9. A polypeptide fragment, which is selected from the following: (i) a novel florigen polypeptide fragment including an amino acid sequence which includes at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177, in an amino acid sequence set forth in SEQ ID NO: 1; (ii) a novel 14-3-3 protein polypeptide fragment including an amino acid sequence which includes at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256, in an amino acid sequence set forth in SEQ ID NO: 2; and (iii) a novel bZIP transcription factor polypeptide fragment including an amino acid sequence which includes at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195, in OsFD1 set forth in SEQ ID NO: 3. 10. A polynucleotide, which encodes any one of the polypeptide fragments (i) to (iii) of the above-mentioned item 9. 11. A florigen activation complex, including a florigen polypeptide fragment bound to a bZIP transcription factor polypeptide fragment via a 14-3-3 protein polypeptide fragment,

in which the florigen polypeptide fragment, the 14-3-3 protein polypeptide fragment, and the bZIP transcription factor polypeptide fragment include the polypeptide fragments (i) to (iii) of the above-mentioned item 9, respectively.

12. A crystal of a florigen activation complex, including a florigen bound to a bZIP transcription factor via a 14-3-3 protein. 13. A crystal of a florigen activation complex according to the above-mentioned item 12, in which the crystal has a space group of P1, P6522, or P4 and lattice constants of a=74 to 158 Å, b=64 to 158 Å, c=96 to 500 Å, α=66 to 90°, β=85 to 90°, and γ=75 to 120°. 14. A method of producing the crystal of a florigen activation complex according to the above-mentioned item 12 or 13, the method including the steps of:

crystallizing a solution containing a complex of a florigen and a 14-3-3 protein by a vapor diffusion method using a precipitant solution containing at least as a precipitant 5 to 35 vol % polyethylene glycol;

collecting the resultant crystal; and

obtaining a crystal of a florigen activation complex by incubating the resultant crystal using a precipitant solution containing at least as a precipitant 5 to 35 vol % polyethylene glycol and containing a bZIP transcription factor.

15. A method of screening a substance that regulates flowering of a plant by designing and/or selecting a candidate substance having a function of regulating an activity of a florigen activation complex using a computer, the method including the steps of: (a) causing storage means to store conformational information obtained from the crystal of a florigen activation complex of the above-mentioned item 12 or 13; (b) causing deriving means to derive a three-dimensional conformation model based on the conformational information; (c) causing calculation means to calculate an interatomic distance in the derived three-dimensional conformation model; and (d) causing calculation means to calculate, based on the calculated interatomic distance, conformational information on a candidate substance capable of enhancing and/or inhibiting binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor, and to design and/or select the candidate substance. 16. A transformant, in which binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor are/is promoted and/or suppressed, the transformant having at least one of the polynucleotide of the above-mentioned item 10 introduced therein so that the polynucleotide is capable of being expressed. 17. A crystal of a florigen activation complex according to the above-mentioned item 12 or 13,

in which:

the florigen includes a polypeptide including an amino acid sequence which includes at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177, in an amino acid sequence set forth in SEQ ID NO: 1;

the 14-3-3 protein includes a polypeptide including an amino acid sequence which includes at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256, in an amino acid sequence set forth in SEQ ID NO: 2; and

the bZIP transcription factor includes a polypeptide including an amino acid sequence which includes at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195, in an amino acid sequence set forth in SEQ ID NO: 3.

18. A crystal of a florigen activation complex according to any one of the above-mentioned items 12, 13, and 17, in which the florigen includes a polypeptide including a sequence of amino acids at positions 6 to 170 in the amino acid sequence set forth in SEQ ID NO: 1, the 14-3-3 protein includes a polypeptide including a sequence of amino acids at positions 1 to 235 in the amino acid sequence set forth in SEQ ID NO: 2, and the bZIP transcription factor includes a polypeptide including a sequence of amino acids at positions 187 to 195 in the amino acid sequence set forth in SEQ ID NO: 3. 19. A crystal of a florigen activation complex according to any one of the above-mentioned items 12, 13, 17, and 18, in which the crystal is selected from the following: (1) a crystal having a space group of P1 and lattice constants of a=74 to 79 Å, b=94 to 99 Å, c=96 to 101 Å, α=66 to 70°, β=85 to 90°, and γ=75 to 79°; (2) a crystal having a space group of P6522 and lattice constants of a=125 to 135 Å, b=125 to 135 Å, c=340 to 344 Å, α=90°, β=90°, and γ=120°; and (3) a crystal having a space group of P4 and lattice constants of a=153 to 158 Å, b=153 to 158 Å, c=495 to 498 Å, α=90°, β=90°, and γ=90°. 20. A crystal of a florigen activation complex according to any one of the above-mentioned items 12, 13, and 17 to 19, in which the crystal is selected from the following: (1) a crystal having a space group of P1 and lattice constants of a=76.7 Å, b=96.6 Å, c=99.5 Å, α=68.2°, β=87.9°, and γ=77.9°; (2) a crystal having a space group of P1 and lattice constants of a=76.8 Å, b=97.3 Å, c=99.8 Å, α=68.1°, β=87.8°, and γ=77.9°; (3) a crystal having a space group of P1 and lattice constants of a=76.2 Å, b=96.1 Å, c=99.1 Å, α=68.2°, β=88.6°, and γ=77.8°; (4) a crystal having a space group of P6522 and lattice constants of a=129.0 Å, b=129.0 Å, c=342.0 Å, α=90°, β=90°, and γ=120°; and (5) a crystal having a space group of P4 and lattice constants of a=155.9 Å, b=155.9 Å, c=496.4 Å, α=90°, β=90°, and γ=90°. 21. A method of producing a crystal of a florigen activation complex according to the above-mentioned item 14, in which the concentration of the solution containing a complex of a florigen and a 14-3-3 protein is 5 to 40 mg/mL, the precipitant solution in the step of crystallizing a solution containing a complex of a florigen and a 14-3-3 protein contains 0.05 to 0.1 M HEPES (pH 6.5 to 8.5), 0.01 to 0.4 M ammonium sulfate, and 15 to 30 vol % polyethylene glycol having a molecular weight of 2,000 to 4,000, and the precipitant solution in the step of obtaining a crystal of a florigen activation complex contains 15 to 30 vol % ethylene glycol, a 1 to 5 mM bZIP transcription factor, 0.05 to 0.1 M HEPES (pH 6.5 to 8.5), 0.2 to 1 M ammonium sulfate, and 15 to 30 vol % polyethylene glycol having a molecular weight 2,000 to 4,000. 22. A method of screening a substance that regulates flowering of a plant according to the above-mentioned item 15, in which the conformational information is one described in Table 1 or Table 2. 23. A method of screening a substance that regulates flowering of a plant according to the above-mentioned item 15 or 22, in which a binding site between the florigen and the 14-3-3 protein includes at least one site selected from the group consisting of D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2, and a binding site between the 14-3-3 protein and the bZIP transcription factor includes at least one site selected from the group consisting of K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and R189 to F195 in SEQ ID NO: 3. 24. A method of screening a substance that regulates flowering of a plant according to any one of the above-mentioned items 15, 22, and 23, the method further including the steps of: acquiring the designed or selected candidate substance; and bringing the candidate substance into contact with a florigen, a 14-3-3 protein, and/or a bZIP transcription factor for investigating a function of regulating an activity of a florigen activation complex of the candidate substance. 25. A method of screening a substance that regulates flowering of a plant according to any one of the above-mentioned items 15 and 22 to 24, the method further including the steps of: producing a crystal of a florigen activation complex by the method of producing a crystal of a florigen activation complex according to the above-mentioned item 14 or 21; and obtaining conformational information on the florigen activation complex by subjecting the crystal to X-ray crystallographic analysis.

Advantageous Effects of Invention

The crystal of the present invention provides conformational information on a florigen activation complex important for the elucidation of a mechanism for regulating flowering of plants. In addition, the crystal may be used as a material for additional research on the mechanism for regulating flowering. Further, it is possible to artificially regulate flowering of plants based on the conformational information obtained from the crystal of the present invention. In the present invention, the binding site of each protein in the florigen activation complex has been clarified. Information on the binding site may be used as a novel target of floral regulation, which can contribute to efficient floral regulation. A transgenic plant, which may be widely utilized for an increase of yield of an agricultural product, an improvement in efficiency of breeding, and the like, can be obtained based on the conformational information obtained in the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A view illustrating a conformation of a florigen activation complex including three proteins Hd3a, GF14c, and OsFD1.

FIG. 2 An image showing the results of in vitro GST pull-down assays of three combinations, i.e., Hd3a and OsFD1, Hd3a and GF14c, GF14c and OsFD1 (Example 1(1)).

FIG. 3 A graph showing the results of analysis of an interaction between Hd3a and GF14c using NMR (Example 1(2)).

FIG. 4 Views illustrating the details of an interaction between Hd3a and GF14c. FIG. 4 b is an expanded view illustrating the vicinity of Hd3a R64 (Example 3).

FIG. 5 Images showing the results of an in vitro GST pull-down assay with GF14c using mutant Hd3a (FIG. 5 a) and the results of an in vitro GST pull-down assay with Hd3a using mutant GF14c (FIG. 5 b) (Example 6).

FIG. 6 An image showing the results of a gel-shift assay of a florigen activation complex including Hd3a, GF14c, and OsFD1 and C-box DNA (Example 7).

FIG. 7 An image showing the results of confirmation of an interaction between each isoform of OsGF14 and Hd3a by a yeast two-hybrid method (Example 8).

FIG. 8 Images showing the results of confirmation of interactions among Hd3a, OsGF14b, and OsFD1 by a yeast two-hybrid method (Example 9).

FIG. 9 An image showing the results of confirmation of interactions among Hd3a, OsGF14b, and OsFD1 in the shoot apex of rice by immunoprecipitation (Example 10).

FIG. 10 An image showing the results of confirmation of subcellular localization of Hd3a, OsGF14b, and OsFD1 (Example 11).

FIG. 11 Images showing the results of confirmation of subcellular localization of Hd3a, OsGF14b, and OsFD1 (Example 11).

FIG. 12 Images showing the results of confirmation of subcellular localization of Hd3a, OsGF14b, and OsFD1 (Example 11).

FIG. 13 Graphs showing the results of confirmation of an influence of transient expression of Hd3a, OsGF14b, and OsFD1 on OsMADS15 gene transcription (Example 12).

FIG. 14 An image showing the results of confirmation of an influence of Hd3a mutation on flowering of plants (Example 13-1).

FIG. 15 A view illustrating an action mechanism of a florigen activation complex, which is obtained by the present invention.

FIG. 16 An image showing the results of confirmation of an influence of Hd3a mutation on flowering of plants (Example 13-2).

DESCRIPTION OF EMBODIMENTS

In general, a plant has a vegetative growth stage and a reproductive growth stage and forms flower buds upon the transition of a growth phase from vegetative growth to reproductive growth. This phenomenon of transition from vegetative growth to reproductive growth is referred to as “flowering.” For example, the flowering in rice and wheats as monocotyledonous plants is heading. The heading refers to that the internode (panicle base) below the ear rapidly elongates and appears from the leaf sheath of the flag leaf. The time when the ear tip appears and the time when the ear including the base portion completely appears are defined as the heading in rice and wheats, respectively.

In the present invention, the plant means a higher plant having a florigen, and is preferably a short-day plant, which forms flower buds when sunshine duration per day is equal to or less than a predetermined time period, more preferably a monocotyledonous short-day plant, still more preferably a gramineous plant, most preferably rice.

(Florigen Activation Complex)

The present invention relates to a florigen activation complex including a complex of a florigen, a 14-3-3 protein, and a bZIP transcription factor, in which the florigen is bound to the bZIP transcription factor via the 14-3-3 protein.

In the present invention, the florigen is not particularly limited, and examples thereof include rice Hd3a and RFT1 (Genbank Accession No. AB062676), Arabidopsis FT (Genbank Accession No. AB027504) and TSF (Genbank Accession No. AB027506), tomato SFT (Genbank Accession No. AY186735), and wheat VRN3 (Genbank Accession No. LOC100037541). Of those, rice Hd3a is preferred. Rice Hd3a includes a protein represented by an amino acid sequence set forth in SEQ ID NO: 1 (Os06g0157700) (Genbank Accession No. BAB61028: derived from Oryza sativa Japonica group cultivar Nipponbare), or a protein which is formed of an amino acid sequence having deletions, substitutions, additions, and/or insertions of one to several amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1, and functions as a florigen. Rice Hd3a in the present invention is preferably a florigen polypeptide fragment which includes at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177 in SEQ ID NO: 1, more preferably a polypeptide fragment formed of a sequence of amino acids at positions 6 to 170 in SEQ ID NO: 1. Further, it is particularly preferred that the polypeptide fragment have mutations of C43L/C109S/C166S in SEQ ID NO: 1.

In the present invention, the 14-3-3 protein is not particularly limited but is preferably rice GF14. Rice GF14 has eight isoforms, i.e., OsGF14a (Os08g0480800), OsGF14b (Os04g0462500), OsGF14c (Os08g0430500), OsGF14d (Os11g0546900), OsGF14e (Os02g0580300), OsGF14f (Os03g0710800), OsGF14g (Os01g0209200), and OsGF14h (Os11g0609600). In the present invention, GF14b, GF14c, GF14e, or GF14f is preferred, and GF14c or GF14b is more preferred. It should be noted that, in this description, numbers beginning with Os are numbers of gene loci specified in the Rice annotation project database (RAP-DB), unless otherwise stated. Those numbers may be used for specifying amino acid sequences and base sequences of proteins such as GF14 isoforms.

Rice GF14c includes a protein represented by an amino acid sequence set forth in SEQ ID NO: 2 (Os08g0430500) (Genbank Accession No. AAB07457.1: derived from Oryza sativa Japonica group cultivar Nipponbare), or a protein which is formed of an amino acid sequence having deletions, substitutions, additions, and/or insertions of one to several amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2, and functions as a 14-3-3 protein to bind to a florigen. The 14-3-3 protein in the present invention is preferably a polypeptide fragment which includes at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256 in SEQ ID NO: 2, more preferably a polypeptide fragment formed of a sequence of amino acids at positions 1 to 235 in SEQ ID NO: 2.

Further, rice GF14b includes a protein represented by an amino acid sequence set forth in SEQ ID NO: 4 (Os04g0462500) (Genbank Accession No. AK071822: derived from Oryza sativa Japonica group cultivar Nipponbare), or a protein which is formed of an amino acid sequence having deletions, substitutions, additions, and/or insertions of one to several amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4, and functions as a 14-3-3 protein to bind to a florigen. Rice GF14b has an amino acid sequence shifted by 6 residues as compared to the amino acid sequence of GF14c. The sequence of amino acids at positions 51 to 227 in SEQ ID NO: 2 corresponds to a sequence of amino acids at positions 57 to 233 in SEQ ID NO: 4. The amino acid sequence which starts with one of amino acids at positions 1 to 5 and ends with one of amino acids at positions 230 to 256 in SEQ ID NO: 2 corresponds to an amino acid sequence which starts with one of amino acids at positions 7 to 11 and ends with one of amino acids at positions 236 to 262 in SEQ ID NO: 4.

In the present invention, the bZIP transcription factor is not particularly limited and examples thereof include rice OsFD1 and Arabidopsis FD (Genbank Accession No. AB105823). Of those, rice OsFD1 is preferred. Rice OsFD1 includes a protein represented by an amino acid sequence set forth in SEQ ID NO: 3 (Os09g0540800: derived from Oryza sativa Japonica group cultivar Nipponbare) (Rice genome annotation locus No. LOC_Os09g36910.1 specified in the TIGR Rice Genome Annotation Database), or a protein which is formed of an amino acid sequence having deletions, substitutions, additions, and/or insertions of one to several amino acid residues in the amino acid sequence set forth in SEQ ID NO: 3, and functions as a bZIP transcription factor. The bZIP transcription factor in the present invention is preferably a polypeptide fragment which includes at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195 in SEQ ID NO: 3, more preferably a polypeptide fragment formed of a sequence of amino acids at positions 187 to 195 in SEQ ID NO: 3. Further, in the polypeptide fragment, serine at position 192 is preferably phosphorylated. The phosphorylation of the serine is considered to be necessary for the binding of the 14-3-3 protein to the bZIP transcription factor.

The florigen, the 14-3-3 protein, and the bZIP transcription factor bind to each other to form a florigen activation complex. A region of a sequence of amino acids at positions 62 to 132 in SEQ ID NO: 1 or a region corresponding to the region in the florigen having an amino acid sequence other than SEQ ID NO: 1, and/or a region of a sequence of amino acids at positions 200 to 227 in SEQ ID NO: 2 or a region corresponding to the region in the 14-3-3 protein having an amino acid sequence other than SEQ ID NO: 2 are/is considered to be important for binding between the florigen and the 14-3-3 protein. Further, it has been clarified in the present invention that a region of a sequence of amino acids at positions 51 to 227 in SEQ ID NO: 2 or a region corresponding to the region in the 14-3-3 protein having an amino acid sequence other than SEQ ID NO: 2, and/or a region of a sequence of amino acids at positions 189 to 195 in SEQ ID NO: 3 or a region corresponding to the region in the bZIP transcription factor having an amino acid sequence other than SEQ ID NO: 3 are/is important for binding between the 14-3-3 protein and the bZIP transcription factor.

In addition, among those regions, amino acids of D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 or amino acids corresponding to the amino acids in the florigen having an amino acid sequence other than SEQ ID NO: 1, and/or amino acids of F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2 or amino acids corresponding to the amino acids in the 14-3-3 protein having an amino acid sequence other than SEQ ID NO: 2 are considered to be important for binding between the florigen and the 14-3-3 protein. Further, it has been clarified in the present invention that amino acids of K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 or amino acids corresponding to the amino acids in the 14-3-3 protein having an amino acid sequence other than SEQ ID NO: 2 are important for binding between the 14-3-3 protein and the bZIP transcription factor.

The “site of a protein corresponding to a particular amino acid (e.g., D62) in SEQ ID NO: X” to be used in the present invention means to include, in addition to a site of a particular amino acid (e.g., D62) in SEQ ID NO: X, a site corresponding to the particular amino acid (e.g., D62) in a protein having an amino acid sequence other than SEQ ID NO: X and having a function equivalent to that of a protein of SEQ ID NO: X. For example, the “sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, and R226 in SEQ ID NO: 2” include, in addition to sites of F200, D201, I204, E212, Y215, and R226 in SEQ ID NO: 2, amino acids corresponding to the amino acids in the 14-3-3 protein having an amino acid sequence other than SEQ ID NO: 2, for example, F206, D207, I210, E218, Y221, and R232 of rice GF14b (corresponding to F200, D201, I204, E212, Y215, and R226 of rice GF14c).

The action mechanism of a florigen activation complex in flowering is described (see FIG. 15). A 14-3-3 protein acts as an adapter of a florigen transported from leaves to the shoot apex. When the florigen enters shoot apex cells, the florigen is considered to first bind to the 14-3-3 protein in the cytoplasm. At this stage, OsFD1 has been expressed in the shoot apex cells, and such complex of the florigen and the 14-3-3 protein enters the nucleus from the cytoplasm and binds to a bZIP transcription factor to form a florigen activation complex, which is retained in the nucleus. When the series of processes are initiated, the florigen activation complex is accumulated in the nucleus. After that, the florigen activation complex activates the transcription of a flowering-related gene (e.g., an OsMADS15 gene), which causes floral induction. In such action mechanism, the florigen is trapped by the 14-3-3 protein in the cytoplasm of the shoot apex cells, and the complex of the florigen and the 14-3-3 protein is efficiently transported by the bZIP transcription factor into the nucleus for the transcriptional activation of a gene necessary for floral induction. Further, the transcriptional activation of the flowering-related gene by the florigen activation complex in the present invention is considered to occur through the binding of the florigen activation complex to C-box DNA. C-box DNA is represented by a base sequence of GACGTC (SEQ ID NO: 10) and is present in a promoter region of the flowering-related gene. The florigen activation complex is estimated to form a stable complex on the C-box of the flowering-related gene and activate the transcription of the flowering-related gene.

In the present invention, the florigen, the 14-3-3 protein, the bZIP transcription factor, mutant proteins thereof, a complex of the florigen and the 14-3-3 protein, a complex of the 14-3-3 protein and the bZIP transcription factor, and the florigen activation complex as a complex of the three proteins may be in such a state that they are isolated and purified from cells, tissues, and the like, or may be in such a state that a gene encoding a protein is introduced into host cells such as yeast and Escherichia coli and the protein is expressed in the cells. Further, the present invention also encompasses polynucleotides encoding those proteins. To those proteins, depending on expression systems of host cells, a peptide fragment may be added in such an amount that functions of the proteins are not affected. For example, in the case of using an expression system of Escherichia coli, an Escherichia coli expression plasmid-derived peptide fragment (GPGHM) has been added to the N-terminal of a protein.

The complex of the florigen and the 14-3-3 protein or the complex of the 14-3-3 protein and the bZIP transcription factor may be produced by bringing the two proteins into contact with each other in an isolated and purified state or in a living body. Similarly, the florigen activation complex as a complex of the three proteins may also be produced by bringing the three kinds of proteins or any one of the complexes of two proteins and the remaining protein into contact with each other in an isolated and purified state or in a living body such as cells.

(Production Method for Crystal of Florigen Activation Complex or Florigen)

First, a protein solution is produced. A protein is allowed to be present in a solution formed of a buffer, a salt, a reducing agent, and the like. Any buffer, salt, and reducing agent may be used as long as the conformation of the protein is not affected. Examples of the buffer include 1 to 500 mM Na-HEPES, sodium phosphate, potassium phosphate, and Tris-HCl. Examples of the salt include 1 mM to 1 M sodium chloride, lithium chloride, and magnesium chloride. Examples of the reducing agent include 0.1 to 10 mM β-mercaptoethanol and dithiothreitol (DTT). Further, the protein solution may contain dimethylsulfoxide (DMSO) or ethylene glycol. The solution containing the protein has a pH of 4 to 11, preferably a pH of 6 to 9. Such protein solution or florigen solution may be used for crystallization without any further treatment, or as necessary, a preservative, a stabilizer, a surfactant, or the like is further added to the solution, and the resultant solution may be used for crystallization.

As a method of crystallizing a protein (polypeptide), a general technique for protein crystallization such as a vapor diffusion method, a batch method, or a dialysis method may be employed. Further, in the crystallization of a protein, it is important to determine physical and chemical factors such as the concentration of the protein, the concentration of a salt, a pH, the kind of a precipitant, and a temperature.

The vapor diffusion method refers to a method involving placing a droplet of a protein solution including a precipitant in a container including a buffer (external solution) containing the precipitant at a higher concentration, sealing the container, and then leaving the resultant to stand still. The vapor diffusion method is classified into a hanging drop method and a sitting drop method depending on how to place the droplet, and any of the methods may be adopted in the present invention. The hanging drop method is a method involving placing a small droplet of a protein solution on a cover glass, inverting the cover glass in a reservoir, and sealing the reservoir. On the other hand, the sitting drop method is a method involving installing an appropriate droplet stage in a reservoir, placing a droplet of a protein solution on the droplet stage, and sealing the reservoir with a cover glass or the like. In any of the methods, a precipitant is incorporated into the solution in the reservoir (reservoir solution). As appropriate, a small amount of the precipitant may be incorporated into a protein small droplet.

The reservoir solution (also referred to as precipitant solution) to be used in the vapor diffusion method is a solution formed of a buffer, a precipitant, a salt, and the like. Any buffer, precipitant, and salt may be used as long as a crystal can be efficiently produced. For example, the buffer is selected from 5 to 200 mM Na-HEPES, sodium phosphate, potassium phosphate, Tris-HCl, sodium acetate, citric acid, cacodylic acid, and the like at a pH of 4 to 10, the precipitant is selected from 5 to 35 vol % polyethylene glycol (PEG) having a molecular weight of 550 to 20,000, 0.2 to 2 M ammonium sulfate, 5 to 35 vol % methylpentanediol (MPD), 0.2 to 2 M ammonium tartrate, 5 to 35 vol % isopropanol, and a combination thereof, and the salt is selected from 0.2 to 4 M sodium chloride, lithium chloride, magnesium chloride, and the like. The components for the reservoir solution are not limited to those described above.

A crystal of a florigen activation complex of the present invention may be produced as described below. A florigen and a 14-3-3 protein are mixed with each other and dialyzed against a 1 to 50 mM Tris-HCl buffer (pH 6.5 to 8.5) containing 10 to 50 mM NaCl to prepare a sample of a complex. The protein solution (protein concentration: 5 to 40 mg/mL) is mixed with a precipitant solution (0.05 to 0.1 M HEPES (pH 6.5 to 8.5), 0.01 to 0.4 M ammonium sulfate, and 15 to 30 vol % PEG (molecular weight: 2,000 to 4,000)), and the crystallization of a complex of the florigen and the 14-3-3 protein is performed by a sitting drop method under the condition of 4 to 20° C. After about 1 to 3 weeks, a single crystal of the complex of the florigen and the 14-3-3 protein is obtained. The obtained single crystal is collected and incubated with a precipitant solution (0.05 to 0.1 M HEPES (pH 6.5 to 8.5), 0.01 to 0.4 M (preferably 0.15 to 0.25 M) ammonium sulfate, 15 to 30 vol % (preferably 23 to 27 vol %) PEG (molecular weight: 2,000 to 4,000)) containing 15 to 30 vol % ethylene glycol and a 1 to 5 mM bZIP transcription factor for 10 to 20 minutes. Thus, a crystal of a florigen activation complex suitable for X-ray crystallographic analysis can be obtained.

The crystal of a florigen activation complex of the present invention may be preferably produced as described below. A florigen and a 14-3-3 protein are mixed with each other at a molar ratio of 1:1 to 2 (more preferably 1:1.5) and dialyzed against a 10 mM Tris-HCl buffer (pH 7.5) containing 20 mM NaCl to prepare a sample of a complex. 1 μl of the protein solution (protein concentration: 10 mg/mL) is mixed with 1 μl of a precipitant solution (0.1 M HEPES (pH 7.5), 0.2M ammonium sulfate, and 25 vol % PEG 3350), and the crystallization of a complex of the florigen and the 14-3-3 protein is performed by a sitting drop method under the condition of 4° C. After about 1 to 3 weeks, a single crystal of the complex of the florigen and the 14-3-3 protein is obtained. The obtained single crystal is collected and incubated with a precipitant solution ((0.1 M HEPES (pH 7.5), 0.2 M ammonium sulfate, 25 vol % PEG 3350) containing 25% ethylene glycol and a 2 mM bZIP transcription factor for 10 to 20 minutes (more preferably for 15 minutes). Thus, a crystal of a florigen activation complex can be obtained.

A crystal of a florigen in the present invention may be produced as described below. A purified florigen is dialyzed against a 1 to 50 mM Tris buffer (pH 5.5 to 7.5) containing 10 to 50 mM NaCl and concentrated so as to achieve a concentration of 5 to 40 mg/mL. The resultant protein solution is mixed with a precipitant solution (0.01 to 0.5 M (preferably 0.05 to 0.15 M) cacodylic acid (pH 5.5 to 7.5), 0.01 to 0.5 M (preferably 0.15 to 0.25 M) ammonium tartrate, and 10 to 50 vol % (preferably 25 to 35 vol %) PEG (molecular weight: 5,000 to 12,000), and crystallization is performed by a sitting drop method under the condition of 4 to 20° C. After about 0.5 to 3 days (preferably about 1 day), a crystal of a florigen activation complex suitable for X-ray crystallographic analysis can be obtained.

The crystal of a florigen in the present invention may be preferably produced as described below. A purified florigen is dialyzed against a 10 mM Tris buffer (pH 7.5) containing 20 mM NaCl and concentrated so as to achieve a concentration of 5 mg/ml. 1 μl of the resultant protein solution is mixed with 1 μl of a precipitant solution (0.1 M cacodylic acid (pH 6.5), 0.2 M ammonium tartrate, and 30 vol % PEG 8000), and crystallization is performed by a sitting drop method under the condition of 4° C. After about 0.5 to 3 days (preferably about 1 day), a crystal of a florigen activation complex suitable for X-ray crystallographic analysis can be obtained.

In the present invention, it is preferred to obtain a crystal having such quality as to provide at least a resolution of 10 Å or less, preferably a resolution of 4.0 Å or less, more preferably a resolution of 3.4 Å or less, still more preferably a resolution of 2.8 Å or less, particularly preferably a resolution of 2.4 Å or less when the crystal is subjected to X-ray crystallographic analysis (“Introduction to Protein Structure” Carl Brandon & John Tooze, translated by Yukiteru Katsube et al., Kyoikusha, 1992, pp. 276-277).

(Crystal of Florigen Activation Complex or Florigen)

The crystal of a florigen activation complex or a florigen of the present invention is substantially free of impurities, and has an activity even when dissolved again. Examples of the impurities include a decomposition product of GST, a florigen, or a florigen activation complex and a protein peculiar to Escherichia coli.

The crystal of a florigen activation complex of the present invention has a space group of P1, P6522, or P4 and lattice constants of a=74 to 158 Å, b=64 to 158 Å, c=96 to 500 Å, α=66 to 90°, β=85 to 90°, and γ=75 to 120°. The crystal obtained by the present invention has sufficient quality and size to conduct X-ray crystallographic analysis at a resolution of about 1.0 Å to about 3.5 Å.

The crystal of a florigen activation complex of the present invention is preferably selected from the following crystals:

(1) a crystal having a space group of P1 and lattice constants of a=74 to 79 Å, b=94 to 99 Å, c=96 to 101 Å, α=66 to 70°, β=85 to 90°, and γ=75 to 79°; (2) a crystal having a space group of P6522 and lattice constants of a=125 to 135 Å, b=125 to 135 Å, c=340 to 344 Å, α=90°, β=90°, and γ=120°; and (3) a crystal having a space group of P4 and lattice constants of a=153 to 158 Å, b=153 to 158 Å, c=495 to 498 Å, α=90°, β=90°, and γ=90°.

The crystal of a florigen activation complex of the present invention is more preferably selected from the following crystals:

(florigen activation complex 1) a crystal having a space group of P1 and lattice constants of a=76.7 Å, b=96.6 Å, c=99.5 Å, α=68.2°, β=87.9°, and γ=77.9° at a resolution of 2.4 Å; (florigen activation complex 2) a crystal having a space group of P1 and lattice constants of a=76.8 Å, b=97.3 Å, c=99.8 Å, α=68.1°, β=87.8°, and γ=77.9° at a resolution of 2.2 Å; (florigen activation complex 3) a crystal having a space group of P1 and lattice constants of a=76.2 Å, b=96.1 Å, c=99.1 Å, α=68.2°, β=88.6°, and γ=77.8° at a resolution of 2.8 Å; (florigen activation complex 4) a crystal having a space group of P6522 and lattice constants of a=129.0 Å, b=129.0 Å, c=342.0 Å, α=90°, β=90°, and γ=120° at a resolution of 2.85 Å; and (florigen activation complex 5) a crystal having a space group of P4 and lattice constants of a=155.9 Å, b=155.9 Å, c=496.4 Å, α=90°, β=90°, and γ=90° at a resolution of 2.96 Å.

The crystal of a florigen of the present invention has a space group of P63 and lattice constants of a=65 to 67 Å, b=65 to 67 Å, c=58 to 61 Å, α=90°, β=90°, and γ=120°. The crystal obtained by the present invention has sufficient quality and size to conduct X-ray crystallographic analysis at a resolution of about 1.0 Å to about 3.5 Å (preferably 1.0 to 1.5 Å).

The crystal of a florigen of the present invention is preferably selected from the following crystals:

(florigen 1) a crystal having a space group of P63 and lattice constants of a=65.9 Å, b=65.9 Å, c=59.8 Å, α=90°, β=90°, and γ=120° at a resolution of 1.3 Å; and (florigen 2) a crystal having a space group of P6522 and lattice constants of a=66.0 Å, b=66.0 Å, c=60.2 Å, α=90°, β=90°, and γ=120° at a resolution of 1.4 Å.

Each of those crystals of a florigen are excellent in resolution, and conformational information obtained by subjecting each of those crystals to X-ray structure analysis is suitable for use in docking simulation with a computer or the like.

(X-Ray Crystallographic Analysis)

X-ray crystallographic analysis is most commonly performed as a technique for clarifying a conformation of a protein (polypeptide). This technique involves crystallizing a protein, irradiating the crystal with a monochromatic X-ray, and clarifying conformational information on the protein based on the resultant X-ray diffraction image. The conformational information includes an electron density map and atomic coordinates, and the atomic coordinates may be acquired by analysis according to a method known in the art (D. E. McRee, Practical Protein Crystallography, Academic Press, San Diego (1993)).

The X-ray crystallographic analysis involves the steps of: irradiating a crystal with an X-ray to acquire diffraction data; analyzing the resultant diffraction data to acquire an electron density of a protein (polypeptide); and analyzing the resultant electron density to acquire atomic coordinates of the protein (polypeptide).

In the X-ray crystallographic analysis, through the use of an X-ray diffractometer in a laboratory or a large radiation facility (e.g., ESRF, APS, SPring-8, PF, ALS, CHESS, SRS, LLNL, SSRL, or Brookhaven), diffraction data is collected by oscillation photography or the like with a two-dimensional detector such as an imaging plate or a CCD camera, and an electron density may be obtained from the data to elucidate atomic coordinates.

A crystal of a protein often undergoes damage by irradiation with an X-ray, resulting in a deterioration in diffraction ability. Hence, it is preferred to perform high-resolution X-ray diffraction through low-temperature measurement. The low-temperature measurement refers to a method involving freezing a crystal by rapidly cooling to about −173° C., and collecting diffraction data in the state. In general, in the freezing of a crystal of a protein, a contrivance such as treatment in a solution containing a protectant (cryoprotectant) such as glycerol is made for the purpose of preventing the collapse of the crystal due to the freezing. A frozen crystal may be prepared, for example, by flash freezing involving directly immersing a crystal, which has been immersed in a preservative solution supplemented with a protectant, in liquid nitrogen.

A diffraction image collected by an X-ray diffraction experiment may be processed with data processing software to calculate diffraction intensities obtained by the indexing and integration of individual diffraction spots. Electron densities in a three-dimensional space are derived by performing inverse Fourier transform using the diffraction intensities and phase information of the diffraction spots. In a diffraction experiment, it is impossible in principle to measure phase information on each of the diffraction spots necessary for the calculation of the electron density. Hence, in order to obtain the electron density, the phase as lost information is estimated by a molecular replacement method, a heavy atom isomorphous replacement method, a multiwavelength anomalous dispersion method (MAD method), or a modified method thereof.

An electron density map is depicted based on the thus obtained electron density, and a three-dimensional model is constructed using software which operates in a graphics workstation in accordance with the electron density map. After the construction of the model, structural refinement is performed by a least-squares method or the like to give final atomic coordinates (conformational coordinates) of a protein.

(Conformational Information on Florigen Activation Complex or Florigen)

The atomic coordinates mean mathematical coordinates in which the positions of the atoms of the protein described above are expressed as three-dimensional coordinates. The atomic coordinates substantially mean a space configuration determined depending on distances between the respective molecules (atoms) which construct a chemical structure. When the space configuration is processed on a computer as information, a relative configuration is converted into numerical information as specific coordinates in a certain coordinate system (referred to as conversion to coordinates). This is processing necessary for convenience in performing computer processing, and it should be understood that the nature of the atomic coordinates is a configuration determined depending on distances between the respective molecules (atoms) as described above and is not coordinate values specified temporarily at the time of computer processing. Further, the atomic coordinates as used herein mean coordinates of individual atoms which construct a substance (such as a protein or an amino acid).

In this description, Table 1 shows atomic coordinates of the florigen activation complex 1, and Table 2 shows atomic coordinates of the florigen 1. The data of Tables 1 and 2 are described in conformity with the format of the protein data bank (PDB) (http://www.wwpdb.org/documentation/format23/v2.3.html). Further, in the present invention, through the use of the conformational information represented by the electron density and atomic coordinates, atomic coordinates obtained by homology modeling or the like on a computer may also be obtained as derivatives for a protein having 40% or more homology to the polypeptide of the present invention.

(Method of Screening Substance that Regulates Flowering of Plant)

In a florigen activation complex including a florigen, a 14-3-3 protein, and a bZIP transcription factor, a substance capable of regulating flowering may be obtained from numerous substances by selecting a substance (including a compound) capable of affecting the binding of each protein and a substance (including a compound) having a structure capable of competitively binding to a binding site.

As one aspect of the screening method of the present invention, there is given a method of screening a substance that regulates flowering of a plant by designing and/or selecting a candidate substance having a florigen activation complex activity regulating function using a computer (hereinafter, also simply referred to as “screening method using a computer”). Such screening method involves the following steps of:

(a) causing storage means to store conformational information obtained from the florigen activation complex or crystal of a florigen of the present invention; (b) causing deriving means to derive a three-dimensional conformation model based on the conformational information; (c) causing calculation means to calculate an interatomic distance in the derived three-dimensional conformation model; and (d) causing calculation means to calculate, based on the calculated interatomic distance, conformational information on a candidate substance capable of enhancing and/or inhibiting binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor, and to design and/or select the candidate substance.

Atomic coordinates of a binding site of each protein may be used as the conformational information on the florigen activation complex or the like, and the whole atomic coordinates of each protein, derivatives thereof including the binding site, and parts thereof may be utilized. Further, atomic coordinates of a binding site appropriately altered on a computer so as to become suitable for screening may be utilized in the present invention.

In the step (a) and the step (b), modes of three-dimensional chemical interactions among various proteins may be displayed in detail by inputting atomic coordinates out of the conformational information on the florigen activation complex or the florigen to a computer or a storage medium of the computer in which a computer program that displays atomic coordinates of a molecule operates. There are known a large number of commercially available computer programs that display atomic coordinates of a molecule. In general, those programs include means for inputting atomic coordinates of a molecule, means for deriving a three-dimensional conformation model based on conformational information by deriving means and visually displaying the coordinates on a computer screen, means for measuring or calculating a distance, a bond angle, and the like between atoms in the displayed molecule, means for additionally correcting the coordinates, and the like. In addition, it is also possible to use a program including means for calculating structural energy of a molecule based on coordinates of the molecule and means for calculating free energy in consideration of a solvent molecule such as a water molecule. Computer programs InsightII and QUANTA commercially available from Accerlys are suitably used for the screening method of the present invention. However, computer programs to be used in the present invention are not limited to the above-mentioned programs.

The candidate substance may be any of known and novel substances, and structures, origins, physical properties, and the like thereof are not particularly limited. Further, the candidate substance may be any of a natural compound, a synthetic compound, a high-molecular-weight compound, a low-molecular-weight compound, a peptide, and a nucleic acid analog. A known program has only to be used for the conversion of the conformation of the candidate substance into coordinates. For example, as a program that converts the conformation of the low-molecular-weight compound into coordinates, CORINA (http://www2.chemie.uni-erlangen.de/software/corina/index.html), Concord (http://www.tripos.com/sciTech/in SilicoDisc/chemInfo/concord.html), Converter, or the like may be utilized.

The steps (c) and (d) include the stage of evaluating the matching state of the atomic coordinates of the candidate substance and atomic coordinates having binding sites of a florigen, a 14-3-3 protein, and a bZIP transcription factor (or a complex of two out of the proteins or a florigen activation complex) by overlapping both the coordinates in the same coordinate system, or the stage of calculating an interatomic distance based on the atomic coordinates of the florigen activation complex or the like to design a candidate substance based on the interatomic distance. Those stages may be performed using the above-mentioned commercially available package software and a computer system capable of operating the software. The computer system appropriately includes various means necessary for operating software of interest, for example, storage means for storing a structural formula of a substance such as a compound, means for converting a conformation of a substance such as a compound into coordinates, storage means for storing atomic coordinates of a substance such as a compound, storage means for storing atomic coordinates of each protein to be used in the step (a), storage means for storing evaluation results, means for displaying contents in each storage means, input means such as a keyboard, display means such as a display, and a central processing unit.

Any software for analysis may be used as long as the software can perform an operation for docking a candidate substance to a protein on a computer, and for example, DOCK, FlexX (Tripos), LigandFit (Accelrys), Ludi (Accelrys), and the like may be used. In addition, the operation may be performed interactively using molecular display software such as InsightII. In that case, as an indicator in evaluating the matching state using each of those programs, a free energy calculated value for the whole complex, an empirical scoring function, shape complementarity evaluation, and the like may be arbitrarily selected and used. The indicator allows whether the binding is good or bad to be objectively evaluated.

The design or selection of a substance capable of regulating flowering using atomic coordinates of various proteins such as a florigen and a florigen activation complex allows quick screening on a computer. Further, it is desired to experimentally evaluate a group of candidate substances selected by screening utilizing a computer.

In the method of screening a substance that regulates flowering of a plant using a computer, in order to experimentally evaluate a function of regulating an action of a florigen activation complex of the candidate substance, the candidate substance is preferably synthesized or acquired. The candidate substance has only to be synthesized using a known technique or acquired, for example, by purifying a substance derived from a living body.

In addition, the resultant candidate substance is experimentally evaluated by subjecting the substance to, for example, a biochemical technique or a biological technique using various proteins such as a florigen, which makes it possible to select a more effective substance having a function of regulating an action of a florigen activation complex, and further, a substance that regulates flowering.

As another aspect of the screening method of the present invention, there is given a method itself of selecting a substance having a function of regulating an action of a florigen activation complex (i.e., a substance having a function capable of regulating flowering) using a biochemical technique or a biological technique (hereinafter, also simply referred to as “screening method using a biochemical technique or the like”). In order to confirm whether or not the candidate substance exhibits a function of regulating an action of a florigen activation complex, it is recommended to examine whether or not there is a difference in action of a florigen activation complex, e.g., whether or not there is a difference in amount of a florigen activation complex between the cases where the candidate substance is added and is not added to a system in which the function of regulating an action of a florigen activation complex can be confirmed. The system in which the function of regulating an action of a florigen activation complex can be confirmed is exemplified by the step of bringing the candidate substance into contact with a florigen, a 14-3-3 protein, and/or a bZIP transcription factor, more specifically the following steps:

(1) a step including bringing a candidate substance into contact with any one of a florigen and a 14-3-3 protein, and bringing the candidate substance into contact with any one of the 14-3-3 protein and the florigen, respectively; and (2) a step including bringing a candidate substance into contact with any one of a 14-3-3 protein to which a florigen is bound or unbound and a bZIP transcription factor, and bringing the candidate substance into contact with any one of the bZIP transcription factor and the 14-3-3 protein to which a florigen is bound or unbound, respectively.

The function of regulating an action of a florigen activation complex preferably means a function of enhancing and/or suppressing the formation of a florigen activation complex. It is considered that the enhancement and/or suppression of the formation of a florigen activation complex allows flowering of a plant to be regulated, that is, the flowering to be accelerated and/or delayed.

The step of bringing a candidate substance into contact with a florigen, a 14-3-3 protein, and/or a bZIP transcription factor may be performed by performing a yeast two-hybrid method, a BiFC method, or the like in the presence or absence of the candidate substance to confirm an interaction, and the function of regulating an action of a florigen activation complex may be evaluated. Alternatively, the function of regulating an action of a florigen activation complex may also be evaluated by fixing a florigen and a bZIP transcription factor on a plate, adding a fluorescence-labeled 14-3-3 protein and a candidate substance, and measuring the fluorescence intensity of the plate. In addition, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or the like may be employed for evaluating the function of regulating an action of a florigen activation complex.

In the method of selecting a substance having a function of regulating an action of a florigen activation complex (further, a substance that regulates flowering), it is preferred to use, as an indicator, binding in a binding site between a florigen and a 14-3-3 protein and/or a binding site between a 14-3-3 protein and a bZIP transcription factor. The use of the binding in each of those binding sites as an indicator means confirming the binding state of a binding site in a florigen activation complex. When binding is inhibited in the presence of a candidate substance as compared to the case in the absence of the candidate substance, the candidate substance may be selected as a substance having a function of suppressing the formation of a florigen activation complex. When binding is promoted, the candidate substance may be selected as a substance having a function of enhancing the formation of a florigen activation complex.

The binding state of a binding site in a complex may be confirmed by an NMR method, a fluorescence labeling method, or the like.

In the NMR method, for example, in the case of binding an unlabeled 14-3-3 protein and bZIP transcription factor to a stable isotope-labeled florigen in the presence of a candidate substance, when the NMR spectrum of the florigen changes in a binding site-specific manner as compared to the case in the absence of the candidate substance, it can be confirmed that the candidate substance affected binding in the binding site. When NMR signals from the florigen have been assigned for all residues (for example, when the florigen is Hd3a, which amino acid of Hd3a gives each NMR peak has been identified), an amino acid to which the candidate substance is bound can be specified.

In the fluorescence labeling method, for example, an amino acid in the vicinity of a binding site is mutated into a cysteine residue and subjected to a reaction with a fluorescence reagent which acts in a cysteine-specific manner to introduce a fluorescence label, and the resultant may be used. When the binding state changes depending on the presence or absence of a candidate substance, it is considered that the intensity of the introduced fluorescence also changes. Thus, the binding state of the binding site can be confirmed.

The NMR method may be performed by dissolving a florigen, a 14-3-3 protein, and a bZIP transcription factor in a solution and measuring the NMR spectrum of the solution. Any solution may be used for dissolving the florigen and the like as long as the NMR spectrum can be measured, and for example, a buffer containing dithiothreitol (DTT), potassium chloride (KCl), sodium chloride (NaCl), and deuterium oxide is used. As an NMR measurement method, homonuclear multidimensional NMR measurement, heteronuclear multidimensional NMR measurement, or the like is preferably employed. For example, the measurement may be performed by an NMR measurement method called 1H-15NHSQC. Such measurement is a technology known to a person skilled in the art. 1H-15NHSQC is a correlation spectrum of a hydrogen atom and a nitrogen atom in a peptide bond in a protein, that is, a 1H-15N correlation spectrum, and information on individual residues may be obtained from a 1H-15N signal attributed to a main chain. Such NMR measurement method allows the conformation analysis of a target high-molecular-weight substance such as a protein, and allows the interaction analysis of a protein.

In the screening method of the present invention, a screening method using a computer and a screening method using a biochemical technique or the like may be employed in combination.

In addition, the evaluation of a function of regulating flowering of a plant, that is, a function of accelerating and/or delaying of flowering, of a candidate substance may be examined by feeding the candidate substance to a plant body (e.g., the candidate substance is absorbed with water from the root, or a transformant is produced by introducing a gene or the like encoding the candidate substance).

(Method of Regulating Flowering of Plant)

The present invention also encompasses a method of regulating flowering of a plant. The regulation of flowering means accelerating and/or delaying flowering. In more detail, the present invention relates to a method of regulating flowering of a plant, the method including promoting (enhancing) and/or suppressing the formation of a florigen activation complex by affecting any one or a plurality of binding sites shown in the following items (1) and/or (2), that is, by regulating binding in the binding sites:

(1) binding sites between a florigen and a 14-3-3 protein: sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and (2) binding sites between a 14-3-3 protein and a bZIP transcription factor: sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO: 3.

The promotion (enhancement) and/or suppression of the formation of a florigen activation complex is exemplified by the introduction of a mutation into a binding site between a florigen and a 14-3-3 protein and/or a binding site between a 14-3-3 protein and a bZIP transcription factor, and the feeding of a substance capable of promoting and/or suppressing binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor to a plant body.

Examples of the substance capable of promoting binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor include a gene itself encoding the florigen, the 14-3-3 protein, or the bZIP transcription factor. Examples of the gene encoding the florigen, the 14-3-3 protein, or the bZIP transcription factor include genes encoding rice Hd3a (SEQ ID NO: 11), rice GF14b (SEQ ID NO: 14), rice GF14c (SEQ ID NO: 12), and rice FD1 (SEQ ID NO: 13). The gene encoding the florigen, the 14-3-3 protein, or the bZIP transcription factor may be a gene encoding a protein (mutant protein) having a mutation in at least one binding site among sites corresponding to D62 and the like in SEQ ID NO: 1, sites corresponding to F200 and the like in SEQ ID NO: 2, and sites corresponding to R189 to F195 in SEQ ID NO 3. A gene encoding a mutant protein may be produced by introducing a mutation using a genetic engineering technique well-known to a person skilled in the art such as a site-directed mutagenesis method. For example, a gene encoding a mutant protein may be produced by combining a PCR reaction, a restriction enzyme reaction, a ligation reaction, and the like. Specifically, a kit such as a QuikChange™ Site-Directed Mutagenesis Kit (STRATAGENE) may be used. The (over)expression of each of those genes in a plant body, preferably in a desired plant tissue cells, more preferably in cell organelles allows binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor to be promoted, and allows flowering to be promoted (accelerated).

A method of (over) expressing a gene is not particularly limited. In general, however, there is adopted a known method such as introducing an isolated gene into plant cells in an expressible manner by a genetic engineering technique. The known method is exemplified by a method involving introducing a recombinant vector containing a desired gene into a host. Specific examples thereof may include a method utilizing infection with Agrobacterium bacteria, such as a protoplast co-culture method or a leaf disk method, a polyethylene glycol method, an electroporation method, a microinjection method, a particle gun method, a liposome method, and an introduction method using an appropriate vector system, and an optimum method has only to be selected from the methods depending on the kind of host cells. It should be noted that examples of the vector include, but not particularly limited to, a plasmid, a phage, and a cosmid, and the vector has only to be appropriately selected depending on the kind of host cells. Further, a wide range of kinds of plants may be used as the host cells. In addition, the host cells may be proliferative plant materials such as protoplasts, cells, calli, organ leaves, seeds, germs, pollens, egg cells, and zygotes, or may be parts of a plant body such as flowers, fruits, leaves, roots, or rooted cuttings.

The promotion of flowering means shortening a vegetative growth period and accelerating the formation of flowers. It is considered that this allows picking seasons of agricultural and horticultural crops to be shifted (generally accelerated), and allows an improvement in efficiency of breeding and increases in yield of agricultural and horticultural crops to be achieved. Further, abilities to form flowers and seeds are estimated to be normal, and hence plant seeds can be quickly produced.

Further, examples of the substance capable of suppressing binding between a florigen and a 14-3-3 protein and/or binding between a 14-3-3 protein and a bZIP transcription factor include florigen, 14-3-3 protein, and bZIP transcription factor polypeptide fragments each having a binding ability, and modified proteins of a florigen and the like, which inhibit functions of normal proteins. The introduction of a gene expressing each of those substances into a plant body allows the formation of a florigen activation complex to be inhibited and allows flowering to be regulated. The “modification” as used herein means completely deleting any one of amino acid sequence regions characteristic of those proteins, and introducing deletions, substitutions, and/or insertions into one or several amino acid residues for a particular site of those sequence regions. The modified protein includes a mutant protein.

A method of modifying a gene encoding each protein has only to be performed by a conventionally known technique and is not particularly limited. That is, as mentioned above, the method has only to be performed, for example, by introducing a mutation into a base sequence utilizing a PCR method, by a known site-directed mutagenesis method (Kunkel et al.: Proc. Natl. Acad. Sci. USA, vol. 82. p 488- (1985)), or with a commercially available kit (e.g., Quikchange Site-Directed Mutagenesis Kit: STRATAGENE).

The modified protein (including a polypeptide) acts in a dominant manner on a protein obtained from a wild-type gene, and suppresses the transcription of a target gene to inhibit the expression of the target gene. Therefore, through the use of a modified gene, a function inherent in a wild-type gene is inhibited and lost, and a transformant in which flowering is suppressed can be produced. Hence, the time and effort required for the production of a transformant in which flowering is suppressed can be reduced.

A method of introducing a modified protein may be performed by the above-mentioned gene expression method.

Further, the introduction of a mutation into a binding site of an endogenous protein of a plant body, and the knockout of a protein may be performed by any of known methods utilizing an antisense method, a gene targeting method, a gene knockout tagging method, and the like (Kempin et al. Nature 389: 802-803. 1997).

The antisense method is a method involving constructing a vector containing an appropriate promoter and a target gene in a reverse direction (antisense DNA) disposed downstream of the promoter, and introducing the vector into a plant, thereby suppressing the expression of the target gene. The gene targeting method is a method involving inserting another DNA by homologous recombination into part of a target gene to knock out the target gene. The method of the present invention using the gene knockout tagging method is a method involving inserting T-DNA or a transposon into a genome at random and screening a target gene-knockout strain by utilizing PCR.

The suppression of flowering means extending a vegetative growth period and delaying the formation of flowers. It is considered that the suppression results in an increase in weight of a plant body as compared to a wild-type plant body (Development 135, 767-774 (2008) doi:10.1242/dev.008631, FIG. 3E). That is, the suppression of flowering allows a plant body having an increased amount of biomass to be produced per individual to be provided. The increased amount of biomass to be produced means that a total weight per individual is large as compared to a wild-type, and also includes the case where the weight of part of tissues of a plant body is specifically large and the weights of the other tissues are similar to those of a wild-type. Plant biomass is generated by fixation of carbon dioxide in air using solar energy, and hence can be trapped as so-called carbon neutral energy. An increase in plant biomass has effects of global environment conservation, global warming prevention, and greenhouse gas emission reduction. Further, for a plant in which parts other than organs involved in flower-bud formation (flowers or seeds) are used as foods and the like, increases in yield of agricultural and horticultural crops per individual can be achieved by increasing the weight of a plant body through the suppression of flowering.

(Transformant)

The present invention also encompasses a transformant having a desired gene introduced therein or a transformant having a desired gene knocked out. The transformant of the present invention includes one obtained in the method of regulating flowering of a plant. The “transformant” includes a cell, a tissue, an organ, and a protoplast as well as a plant individual. Further, organisms as targets of transformation are also not particularly limited and examples thereof may include various microorganisms (including Escherichia coli and the like) and plants. Further, animals and insects may also be used as targets of transformation by selecting promoters and vectors. Further, the transformant as used herein also includes a plant having introduced therein the gene according to the present invention or an offspring of the plant having the same properties as the plant, or a tissue thereof. Such transformant may be produced using the technique described in the above-mentioned “Method of regulating flowering of plant” section.

EXAMPLES

Hereinafter, the present invention is specifically described by way of examples and experimental examples for further understanding of the present invention. However, it should be appreciated that the scope of the present invention is by no means limited by these examples and experimental examples. Further, all of Hd3a, GF14, and FD1 used in the following examples are proteins derived from rice. In the notation of the proteins, each of the proteins may be specified with or without the prefix “Os.” However, all of the proteins used in the following examples are proteins derived from rice.

First, a production method for a plasmid used in examples is shown below.

Full-length cDNAs for Hd3a (Os06g0157700), OsGF14 (OsGF14a: Os08g0480800, OsGF14b: Os04g0462500, OsGF14c: Os08g0430500, OsGF14d: Os11g0546900, OsGF14e: Os02g0580300, OsGF14f: Os03g0710800, OsGF14g: Os01g0209200, and OsGF14h: Os11g0609600), and OsFD1 (Os09g0540800) were cloned by RT-PCR. The coding regions were PCR-amplified by a conventional method and introduced into a pENTR/D-TOPO cloning vector (Invitrogen) to obtain entry clones.

The introduction of amino-acid substitutions or deletions into OsGF14, OsFD1, and Hd3a was performed by PCR with KOD FX DNA polymerase (TOYOBO). The PCR-amplified fragments were introduced into the pENTR/D-TOPO cloning vector to obtain entry clones.

pGII pUbq GW-T7 and pGII pUbq HA-GW were produced by inserting a ubiquitin promoter derived from maize, an NOS termination region (Miki and Shimamoto, Plant Cell Physiol. 45, 490 (2004).), and an attR recombination region (Nakagawa et al., 2007) with a T7 tag region or an HA tag region into a pGreen II vector (Hellens et al., Plant Mol. Biol. 42, 819 (2000).).

A pUbq:Hd3a-mCherry expression vector (Non Patent Literature 2) and pHd3a:Hd3a-GFP and prolC:Hd3a-GFP binary vectors (Tamaki et al., Science 316, 1033 (2007)) were produced based on the descriptions of the literatures. A prolC:Hd3a (R64G)-GFP binary vector and a prolC:Hd3a (R64G/R132A)-GFP binary vector were produced using mutant Hd3a cDNA as described in Tamaki et al., Science 316, 1033 (2007). In order to produce other vectors, a mutant Hd3a gene, a mutant OsGF14 gene, and a mutant OsFD1 gene were transformed into various vectors in pENTR D-TOPO vectors by a Gateway recombination method with Gateway BP clonase II (Invitrogen).

Those vectors may be included in pBTM116-GW and pVP16-GW in a yeast two-hybrid assay (Examples 8 and 9). Further, the vectors may be included in pGII pUbq GW-T7, pGII pUbq HA-GW, p35S-GFP-GW, p35S-mCerulean-GW, p35S-Vn-GW, p35S-Vc-GW, p35S-GW-Vn, p35S-GW-Vc, and pUbq-GW in a transient expression assay (Examples 11 and 12) and in a p2K-GW binary vector and a pANDA RNAi vector (Miki and Shimamoto 2004) in the production of a transgenic plant (Examples 10, 13-1, and 13-2. SV40 NLS was fused with GF14b and GF14e by PCR.

Example 1 Confirmation of Interactions Among Three Proteins

(1) Interactions among three proteins were confirmed by an in vitro GST pull-down assay.

A plasmid having incorporated therein cDNA encoding full-length Hd3a (residues 1 to 179) set forth in SEQ ID NO: 1 and cDNA encoding full-length GF14c (residues 1 to 256) set forth in SEQ ID NO: 2 was subjected to restriction enzyme treatment, and fragments obtained by the restriction enzyme treatment were fused with a polynucleotide encoding GST and introduced into a vector. The vector was introduced into Escherichia coli, and a GST-fused GF14c protein or a GST-fused Hd3a protein was expressed in Escherichia coli. After the culture of Escherichia coli, Escherichia coli was harvested by centrifugation, and the GST-fused GF14c protein or the GST-fused Hd3a protein was isolated and purified from the lysis solution.

10 nmol of each of the isolated and purified GST-fused GF14c protein and GST-fused Hd3a protein were incubated with 10 μl of a Glutathione Sepharose 4B resin (GE Healthcare) to bind each of the polypeptides to the resin. After that, the resin was washed with a phosphate buffer (pH 6.8) containing 50 mM KCl and 1 mM DTT to remove a free GST-fused protein unbound to the resin. The isolated and purified GST protein bound to the resin was used as a negative control. To three kinds of resins to which the GST-GF14c protein, the GST-Hd3a protein, and the GST protein were bound, 1 nmol of each of the isolated and purified proteins (OsFD1 having introduced therein a mutation of S192E in a region of positions 147 to 195 in SEQ ID NO: 3 and full-length Hd3a, both of which were not fused with GST) was added and the mixtures were incubated at room temperature for 15 minutes. After that, the resins were washed with a phosphate buffer (pH 6.8) containing 50 mM KCl and 1 mM DTT to remove a free GST-GF14c polypeptide unbound to the resins. Each of the bound proteins was eluted with a phosphate buffer (pH 6.8) containing 50 mM glutathione, 50 mM KCl, and 1 mM DTT and confirmed by SDS polyacrylamide electrophoresis.

FIG. 2 shows the results. No interaction was observed between Hd3a and OsFD1 (FD1), whereas interactions were observed between Hd3a and GF14c and between GF14c and OsFD1, suggesting that Hd3a and OsFD1 were bound via GF14c.

(2) The interaction between Hd3a and GF14c was confirmed using NMR. A 15N stable isotope-labeled Hd3a protein solution was prepared with a 50 mM phosphate buffer (pH 6.8) containing 100 mM KCl, 1 mM DTT, and 7% deuterium oxide so as to achieve a final concentration of 0.2 mM. After that, 15N-HSQC NMR spectra of the following solutions were measured: the prepared Hd3a protein solution alone; and a mixed solution obtained by adding to the Hd3a protein solution a GF14c protein unlabeled with a stable isotope at a molar ratio of 1:0.5. The spectra were compared to each other. The NMR measurement was performed at a temperature of 30° C. and performed using an AV500 NMR apparatus manufactured by Bruker.

FIG. 3 shows the results. The NMR spectrum of Hd3a changed through binding with GF14c. Thus, binding between Hd3a and GF14c was confirmed. Further, the binding was found to be site-specific because the shift of NMR signals was partially (e.g., Hd3a R64 and M63 in the expanded view of FIG. 3) observed.

Example 2 Production of Crystals of Florigen Activation Complex

A plasmid having incorporated therein cDNA encoding residues 6 to 170 in full-length Hd3a (residues 1 to 179) set forth in SEQ ID NO: 1 and cDNA encoding residues 1 to 235 in full-length GF14c (residues 1 to 256) set forth in SEQ ID NO: 2 was subjected to restriction enzyme treatment, and fragments obtained by the restriction enzyme treatment were fused with a polynucleotide encoding GST and introduced into a vector. The vector was introduced into Escherichia coli. A GST-fused Hd3a protein and GF14c protein were expressed in Escherichia coli. After the culture of Escherichia coli, Escherichia coli was harvested by centrifugation, and the proteins were purified from the lysis solution. After that, analysis was performed by an SDS-polyacrylamide electrophoresis method (SDS-PAGE). As a result, the proteins were each found to have a purity of 95% or more. A polypeptide encoding residues 187 to 195 in full-length OsFD1 (residues 1 to 195) set forth in SEQ ID NO: 3 was prepared by chemical synthesis.

The resultant Hd3a and GF14c were mixed with each other at a molar ratio of 1:1.5 and dialyzed against a 10 mM Tris-HCl buffer (pH 7.5) containing 20 mM NaCl to prepare a sample of a complex. The crystallization of the complex was performed at 4° C. by a sitting drop method using a mixture of 1 μl of the protein solution (protein concentration: 10 mg/mL) and 1 μl of a precipitant solution (0.1 M HEPES (pH 7.5), 0.2 M ammonium sulfate, and 25% PEG 3350). After about 2 weeks, single crystals each measuring 0.1 by 0.1 mm were obtained. The obtained Hd3a-GF14c complex crystals were collected and incubated with the precipitant solution containing 25% ethylene glycol and a 2 mM OsFD1 polypeptide for 15 minutes to produce crystals of a florigen activation complex of Hd3a, GF14c, and OsFD1.

The resultant crystals were the following five kinds:

(florigen activation complex 1) a crystal having a space group of P1 and lattice constants of a=76.7 Å, b=96.6 Å, c=99.5 Å, α=68.2°, β87.9°, and γ=77.9° at a resolution of 2.4 Å; (florigen activation complex 2) a crystal having a space group of P1 and lattice constants of a=76.8 Å, b=97.3 Å, c=99.8 Å, α=68.1°, β87.8°, and γ=77.9° at a resolution of 2.2 Å; (florigen activation complex 3) a crystal having a space group of P1 and lattice constants of a=76.2 Å, b=96.1 Å, c=99.1 Å, α=68.2°, β=88.6°, and γ=77.8° at a resolution of 2.8 Å; (florigen activation complex 4) a crystal having a space group of P6522 and lattice constants of a=129.0 Å, b=129.0 Å, c=342.0 Å, α=90°, β=90°, and γ=120° at a resolution of 2.85 Å; and (florigen activation complex 5) a crystal having a space group of P4 and lattice constants of a=155.9 Å, b=155.9 Å, c=496.4 Å, α=90°, β=90°, and γ=90° at a resolution of 2.96 Å.

Example 3 X-Ray Structure Analysis of Crystals of Florigen Activation Complex

The resultant crystals were measured for their X-ray diffraction images using a CCD detector in the BL5A beamline of a synchrotron radiation research facility Photon Factory. Atomic coordinates of a florigen activation complex were obtained based on the resultant diffraction images.

X-ray diffraction data was collected to perform the indexing of individual diffraction spots and the calculation of diffraction intensities. Phase angles were determined from the resultant diffraction intensities and search models by a molecular replacement method. Electron density maps were derived by inverse Fourier transform based on the diffraction intensities of the diffraction spots and the phase angles described above. Atomic coordinates were constructed based on the resultant electron density maps.

Specifically, the resultant data was processed using HKL2000 and scaled with SCALEPACK. As a search model, the crystal structure of a florigen was determined by a molecular replacement method. The resultant model was refined at 2.4 Å using CNS and REFMAC. After each refinement, the resultant model was corrected with an electron density map 2Fo-Fc map using COOT.

Table 1 shows atomic coordinates obtained from the crystal of the florigen activation complex 1. Further, FIGS. 1 and 4 illustrate conformation models constructed from the atomic coordinates. It is understood that phosphorylated 5192 (pS192) in OsFD1 is specifically recognized by GF14c.

Lengthy table referenced here US20130019345A1-20130117-T00001 Please refer to the end of the specification for access instructions.

Example 4 Production of Crystals of Florigen (1) Production of Expression Vector

A plasmid having incorporated therein cDNA encoding residues 6 to 170 in full-length Hd3a (residues 1 to 179) set forth in SEQ ID NO: 1 was subjected to restriction enzyme treatment. Fragments obtained by the restriction enzyme treatment were fused with a polynucleotide encoding GST and introduced into a vector. The vector was introduced into Escherichia coli. A GST-fused Hd3a protein was expressed in Escherichia coli. After the culture of Escherichia coli, Escherichia coli was harvested by centrifugation, and the Hd3a protein was purified from the lysis solution. After that, analysis was performed by a SDS-polyacrylamide electrophoresis method (SDS-PAGE). As a result, the Hd3a protein was found to have a purity of 95% or more.

The purified Hd3a protein was dialyzed against a 10 mM Tris buffer (pH 7.5) containing 20 mM NaCl and concentrated so as to achieve a concentration of 5 mg/ml. Crystallization was performed at 4° C. by a sitting drop method using a mixture of 1 μl of the protein solution and 1 μl of a precipitant solution (0.1 M cacodylic acid (pH 6.5), 0.2 M ammonium tartrate, and 30% PEG 8000). Single crystals each measuring 0.1 by 0.1 mm were obtained after about 1 day.

The resultant crystals were the following two kinds:

(florigen 1) a crystal having a space group of P63 and lattice constants of a=65.9 Å, b=65.9 Å, c=59.8 Å, α=90°, β=90°, and γ=120° at a resolution of 1.3 Å; and (florigen 2) a crystal having a space group of P6522 and lattice constants of a=66.0 Å, b=66.0 Å, c=60.2 Å, α=90°, β=90°, and γ=120° at a resolution of 1.4 Å.

Example 5 X-Ray Structure Analysis of Crystals of Florigen

The resultant crystals were measured for their X-ray diffraction images using a CCD detector in the BL5A beamline of a synchrotron radiation research facility Photon Factory. Atomic coordinates of a florigen were obtained based on the resultant diffraction images.

X-ray diffraction data was collected to perform the indexing of individual diffraction spots and the calculation of diffraction intensities. Phase angles were determined from the resultant diffraction intensities and search models by a molecular replacement method. Electron density maps were derived by inverse Fourier transform based on the diffraction intensities of the diffraction spots and the phase angles described above. Atomic coordinates were constructed based on the resultant electron density maps.

Specifically, the resultant data was processed using HKL2000 and scaled with SCALEPACK. As a search model, the crystal structure of a florigen was determined by a molecular replacement method. The resultant model was refined at 1.3 Å and 1.4 Å using CNS and REFMAC. After each refinement, the resultant model was corrected with an electron density map 2Fo-Fc map using COOT.

Table 2 below shows atomic coordinates obtained from the crystal of the florigen 1.

TABLE 2 Atomic coordinates of florigen ATOM 1 N GLY A 5 8.706 4.683 11.752 1.00 46.29 N ATOM 2 CA GLY A 5 9.393 3.368 11.940 1.00 46.24 C ATOM 3 C GLY A 5 10.574 3.541 12.876 1.00 46.01 C ATOM 4 O GLY A 5 10.375 3.941 14.027 1.00 46.10 O ATOM 5 N ARG A 6 11.804 3.261 12.435 1.00 45.65 N ATOM 6 CA ARG A 6 12.210 2.730 11.112 1.00 45.26 C ATOM 7 CB ARG A 6 11.465 3.383 9.940 1.00 45.27 C ATOM 8 CG ARG A 6 11.788 2.843 8.535 1.00 45.56 C ATOM 9 CD ARG A 6 11.793 1.306 8.431 1.00 44.76 C ATOM 10 NE ARG A 6 10.455 0.711 8.470 1.00 44.76 N ATOM 11 CZ ARG A 6 10.174 −0.544 8.112 1.00 44.36 C ATOM 12 NH1 ARG A 6 11.133 −1.355 7.673 1.00 43.31 N ATOM 13 NH2 ARG A 6 8.926 −0.989 8.181 1.00 42.93 N ATOM 14 C ARG A 6 13.715 2.991 11.021 1.00 44.86 C ATOM 15 O ARG A 6 14.443 2.270 10.333 1.00 44.69 O ATOM 16 N ALA A 7 14.138 4.063 11.699 1.00 44.40 N ATOM 17 CA ALA A 7 15.500 4.266 12.224 1.00 43.74 C ATOM 18 CB ALA A 7 15.950 3.064 13.070 1.00 43.95 C ATOM 19 C ALA A 7 16.621 4.709 11.283 1.00 43.16 C ATOM 20 O ALA A 7 17.202 3.903 10.552 1.00 43.44 O ATOM 21 N ARG A 8 16.913 6.009 11.342 1.00 42.15 N ATOM 22 CA ARG A 8 18.164 6.599 10.848 1.00 40.76 C ATOM 23 CB ARG A 8 19.375 5.874 11.472 1.00 40.84 C ATOM 24 CG ARG A 8 20.377 6.769 12.212 1.00 40.73 C ATOM 25 CD ARG A 8 21.157 7.706 11.292 1.00 41.25 C ATOM 26 NE ARG A 8 21.484 7.093 10.003 1.00 41.84 N ATOM 27 CZ ARG A 8 22.667 6.576 9.682 1.00 43.45 C ATOM 28 NH1 ARG A 8 22.849 6.045 8.478 1.00 43.18 N ATOM 29 NH2 ARG A 8 23.671 6.582 10.554 1.00 43.26 N ATOM 30 C ARG A 8 18.310 6.740 9.314 1.00 40.13 C ATOM 31 O ARG A 8 19.319 6.320 8.742 1.00 40.05 O ATOM 32 N ASP A 9 17.323 7.356 8.652 1.00 39.15 N ATOM 33 CA ASP A 9 17.456 7.694 7.230 1.00 37.79 C ATOM 34 CB ASP A 9 16.125 8.171 6.626 1.00 38.31 C ATOM 35 CG ASP A 9 14.930 7.384 7.110 1.00 39.78 C ATOM 36 OD1 ASP A 9 13.962 8.033 7.568 1.00 41.40 O ATOM 37 OD2 ASP A 9 14.943 6.135 7.015 1.00 41.38 O ATOM 38 C ASP A 9 18.475 8.823 7.061 1.00 36.45 C ATOM 39 O ASP A 9 18.967 9.371 8.045 1.00 36.65 O ATOM 40 N PRO A 10 18.799 9.184 5.809 1.00 34.65 N ATOM 41 CA PRO A 10 19.433 10.483 5.641 1.00 33.25 C ATOM 42 CB PRO A 10 19.729 10.528 4.142 1.00 32.98 C ATOM 43 CG PRO A 10 19.779 9.089 3.730 1.00 34.18 C ATOM 44 CD PRO A 10 18.692 8.465 4.528 1.00 34.61 C ATOM 45 C PRO A 10 18.500 11.636 6.065 1.00 31.38 C ATOM 46 O PRO A 10 18.947 12.775 6.155 1.00 31.77 O ATOM 47 N LEU A 11 17.225 11.336 6.316 1.00 29.48 N ATOM 48 CA LEU A 11 16.274 12.345 6.813 1.00 27.68 C ATOM 49 CB LEU A 11 14.816 11.916 6.588 1.00 26.63 C ATOM 50 CG LEU A 11 14.223 11.864 5.168 1.00 25.43 C ATOM 51 CD1 LEU A 11 12.770 11.460 5.229 1.00 23.05 C ATOM 52 CD2 LEU A 11 14.346 13.191 4.424 1.00 23.76 C ATOM 53 C LEU A 11 16.500 12.672 8.288 1.00 27.42 C ATOM 54 O LEU A 11 16.226 13.794 8.730 1.00 25.74 O ATOM 55 N VAL A 12 16.976 11.695 9.059 1.00 27.11 N ATOM 56 CA VAL A 12 17.276 11.955 10.468 1.00 27.10 C ATOM 57 CB VAL A 12 17.000 10.742 11.410 1.00 27.47 C ATOM 58 CG1 VAL A 12 16.029 9.764 10.781 1.00 27.69 C ATOM 59 CG2 VAL A 12 18.287 10.049 11.820 1.00 28.05 C ATOM 60 C VAL A 12 18.697 12.474 10.605 1.00 27.02 C ATOM 61 O VAL A 12 18.943 13.378 11.399 1.00 26.52 O ATOM 62 N VAL A 13 19.619 11.920 9.813 1.00 26.34 N ATOM 63 CA VAL A 13 20.974 12.455 9.688 1.00 26.40 C ATOM 64 CB VAL A 13 21.832 11.636 8.672 1.00 26.94 C ATOM 65 CG1 VAL A 13 23.016 12.446 8.154 1.00 27.65 C ATOM 66 CG2 VAL A 13 22.314 10.349 9.295 1.00 27.69 C ATOM 67 C VAL A 13 20.891 13.917 9.239 1.00 25.34 C ATOM 68 O VAL A 13 21.604 14.782 9.758 1.00 25.67 O ATOM 69 N GLY A 14 20.002 14.186 8.279 1.00 24.50 N ATOM 70 CA GLY A 14 19.808 15.539 7.763 1.00 22.95 C ATOM 71 C GLY A 14 18.975 16.440 8.656 1.00 21.25 C ATOM 72 O GLY A 14 18.804 17.619 8.343 1.00 21.51 O ATOM 73 N ARG A 15 18.471 15.890 9.763 1.00 20.71 N ATOM 74 CA ARG A 15 17.691 16.624 10.770 1.00 19.32 C ATOM 75 CB ARG A 15 18.516 17.757 11.419 1.00 19.74 C ATOM 76 CG ARG A 15 19.854 17.337 12.057 1.00 22.06 C ATOM 77 CD ARG A 15 19.672 16.149 12.971 1.00 27.33 C ATOM 78 NE ARG A 15 20.898 15.788 13.677 1.00 31.03 N ATOM 79 CZ ARG A 15 21.086 14.638 14.326 1.00 32.99 C ATOM 80 NH1 ARG A 15 20.134 13.708 14.357 1.00 33.51 N ATOM 81 NH2 ARG A 15 22.236 14.413 14.940 1.00 34.74 N ATOM 82 C ARG A 15 16.344 17.158 10.256 1.00 18.54 C ATOM 83 O ARG A 15 15.694 17.977 10.915 1.00 16.85 O ATOM 84 N VAL A 16 15.926 16.676 9.088 1.00 17.20 N ATOM 85 CA VAL A 16 14.594 16.981 8.554 1.00 16.42 C ATOM 86 CB VAL A 16 14.452 16.497 7.111 1.00 16.16 C ATOM 87 CG1 VAL A 16 13.025 16.678 6.618 1.00 15.31 C ATOM 88 CG2 VAL A 16 15.429 17.228 6.198 1.00 16.22 C ATOM 89 C VAL A 16 13.529 16.339 9.438 1.00 16.42 C ATOM 90 O VAL A 16 12.586 17.007 9.861 1.00 14.92 O ATOM 91 N VAL A 17 13.699 15.042 9.741 1.00 17.05 N ATOM 92 CA VAL A 17 12.971 14.429 10.836 1.00 18.33 C ATOM 93 CB VAL A 17 12.979 12.880 10.776 1.00 18.85 C ATOM 94 CG1 VAL A 17 12.460 12.297 12.090 1.00 20.27 C ATOM 95 CG2 VAL A 17 12.130 12.398 9.592 1.00 19.66 C ATOM 96 C VAL A 17 13.664 14.958 12.096 1.00 18.52 C ATOM 97 O VAL A 17 14.863 14.725 12.292 1.00 19.61 O ATOM 98 N GLY A 18 12.908 15.699 12.902 1.00 18.19 N ATOM 99 CA GLY A 18 13.460 16.496 13.984 1.00 18.34 C ATOM 100 C GLY A 18 13.054 17.950 13.814 1.00 17.75 C ATOM 101 O GLY A 18 12.180 18.441 14.531 1.00 19.12 O ATOM 102 N ASP A 19 13.671 18.636 12.854 1.00 16.90 N ATOM 103 CA ASP A 19 13.386 20.057 12.654 1.00 15.56 C ATOM 104 CB ASP A 19 14.466 20.725 11.810 1.00 15.27 C ATOM 105 CG ASP A 19 15.803 20.825 12.521 1.00 15.22 C ATOM 106 OD1 ASP A 19 15.879 20.650 13.763 1.00 18.15 O ATOM 107 OD2 ASP A 19 16.786 21.090 11.814 1.00 14.86 O ATOM 108 C ASP A 19 12.040 20.309 11.991 1.00 14.92 C ATOM 109 O ASP A 19 11.388 21.298 12.292 1.00 16.18 O ATOM 110 N VAL A 20 11.630 19.418 11.093 1.00 13.95 N ATOM 111 CA VAL A 20 10.422 19.676 10.306 1.00 13.45 C ATOM 112 CB VAL A 20 10.761 19.793 8.804 1.00 12.68 C ATOM 113 CG1 VAL A 20 9.490 20.028 7.991 1.00 11.79 C ATOM 114 CG2 VAL A 20 11.789 20.907 8.550 1.00 14.23 C ATOM 115 C VAL A 20 9.382 18.583 10.489 1.00 13.69 C ATOM 116 O VAL A 20 8.184 18.864 10.607 1.00 13.87 O ATOM 117 N LEU A 21 9.865 17.342 10.540 1.00 14.73 N ATOM 118 CA LEU A 21 8.987 16.190 10.556 1.00 15.26 C ATOM 119 CB LEU A 21 9.166 15.364 9.277 1.00 14.80 C ATOM 120 CG LEU A 21 8.941 16.094 7.945 1.00 12.27 C ATOM 121 CD1 LEU A 21 9.251 15.176 6.794 1.00 12.76 C ATOM 122 CD2 LEU A 21 7.554 16.679 7.823 1.00 12.86 C ATOM 123 C LEU A 21 9.253 15.308 11.752 1.00 16.68 C ATOM 124 O LEU A 21 10.353 15.303 12.306 1.00 16.92 O ATOM 125 N ASP A 22 8.219 14.563 12.111 1.00 17.80 N ATOM 126 CA ASP A 22 8.346 13.448 13.039 1.00 19.01 C ATOM 127 CB ASP A 22 7.083 13.333 13.882 1.00 19.32 C ATOM 128 CG ASP A 22 6.985 14.440 14.926 1.00 20.48 C ATOM 129 OD1 ASP A 22 8.033 14.986 15.317 1.00 24.05 O ATOM 130 OD2 ASP A 22 5.868 14.782 15.330 1.00 23.30 O ATOM 131 C ASP A 22 8.608 12.186 12.239 1.00 20.19 C ATOM 132 O ASP A 22 8.469 12.173 11.001 1.00 20.87 O ATOM 133 N ALA A 23 9.008 11.130 12.941 1.00 20.00 N ATOM 134 CA ALA A 23 9.253 9.829 12.338 1.00 20.49 C ATOM 135 CB ALA A 23 9.538 8.795 13.419 1.00 20.19 C ATOM 136 C ALA A 23 8.059 9.395 11.525 1.00 19.62 C ATOM 137 O ALA A 23 6.915 9.584 11.940 1.00 20.07 O ATOM 138 N PHE A 24 8.347 8.827 10.354 1.00 20.67 N ATOM 139 CA PHE A 24 7.310 8.278 9.475 1.00 21.24 C ATOM 140 CB PHE A 24 6.691 9.382 8.573 1.00 21.46 C ATOM 141 CG PHE A 24 7.599 9.878 7.481 1.00 20.91 C ATOM 142 CD1 PHE A 24 8.475 10.933 7.714 1.00 21.48 C ATOM 143 CE1 PHE A 24 9.318 11.396 6.704 1.00 20.22 C ATOM 144 CZ PHE A 24 9.273 10.814 5.443 1.00 20.95 C ATOM 145 CE2 PHE A 24 8.398 9.772 5.190 1.00 20.11 C ATOM 146 CD2 PHE A 24 7.564 9.308 6.204 1.00 20.15 C ATOM 147 C PHE A 24 7.834 7.104 8.636 1.00 21.22 C ATOM 148 O PHE A 24 9.045 6.933 8.449 1.00 22.17 O ATOM 149 N VAL A 25 6.917 6.306 8.108 1.00 22.54 N ATOM 150 CA VAL A 25 7.291 5.228 7.186 1.00 23.16 C ATOM 151 CB VAL A 25 6.736 3.831 7.637 1.00 23.86 C ATOM 152 CG1 VAL A 25 5.285 3.923 8.093 1.00 25.21 C ATOM 153 CG2 VAL A 25 6.915 2.782 6.548 1.00 24.61 C ATOM 154 C VAL A 25 6.897 5.604 5.749 1.00 22.57 C ATOM 155 O VAL A 25 5.761 5.981 5.494 1.00 23.29 O ATOM 156 N ARG A 26 7.864 5.544 4.841 1.00 22.73 N ATOM 157 CA ARG A 26 7.631 5.866 3.435 1.00 21.98 C ATOM 158 CB ARG A 26 8.945 5.827 2.654 1.00 22.63 C ATOM 159 CG ARG A 26 9.985 6.859 3.069 1.00 22.46 C ATOM 160 CD ARG A 26 11.343 6.518 2.484 1.00 24.06 C ATOM 161 NE ARG A 26 12.366 7.534 2.740 1.00 26.16 N ATOM 162 CZ ARG A 26 13.118 7.594 3.838 1.00 27.74 C ATOM 163 NH1 ARG A 26 12.959 6.708 4.817 1.00 28.99 N ATOM 164 NH2 ARG A 26 14.038 8.541 3.953 1.00 28.55 N ATOM 165 C ARG A 26 6.635 4.870 2.852 1.00 22.09 C ATOM 166 O ARG A 26 6.716 3.663 3.148 1.00 22.31 O ATOM 167 N SER A 27 5.691 5.359 2.046 1.00 21.66 N ATOM 168 CA SER A 27 4.698 4.475 1.413 1.00 20.78 C ATOM 169 CB SER A 27 3.442 4.381 2.269 1.00 21.01 C ATOM 170 OG SER A 27 2.776 5.632 2.331 1.00 22.05 O ATOM 171 C SER A 27 4.315 4.863 −0.021 1.00 21.09 C ATOM 172 O SER A 27 3.502 4.189 −0.659 1.00 21.20 O ATOM 173 N THR A 28 4.878 5.957 −0.513 1.00 20.27 N ATOM 174 CA THR A 28 4.651 6.366 −1.884 1.00 19.84 C ATOM 175 CB THR A 28 3.536 7.430 −1.989 1.00 19.89 C ATOM 176 OG1 THR A 28 2.291 6.839 −1.602 1.00 22.54 O ATOM 177 CG2 THR A 28 3.396 7.989 −3.416 1.00 19.83 C ATOM 178 C THR A 28 5.963 6.912 −2.356 1.00 19.45 C ATOM 179 O THR A 28 6.668 7.586 −1.602 1.00 20.19 O ATOM 180 N ASN A 29 6.308 6.581 −3.590 1.00 18.69 N ATOM 181 CA ASN A 29 7.517 7.079 −4.189 1.00 18.29 C ATOM 182 CB ASN A 29 8.015 6.114 −5.277 1.00 19.37 C ATOM 183 CG ASN A 29 8.370 4.721 −4.709 1.00 20.85 C ATOM 184 OD1 ASN A 29 7.979 3.682 −5.256 1.00 21.94 O ATOM 185 ND2 ASN A 29 9.092 4.707 −3.592 1.00 22.85 N ATOM 186 C ASN A 29 7.349 8.541 −4.637 1.00 17.79 C ATOM 187 O ASN A 29 6.278 8.978 −5.088 1.00 16.90 O ATOM 188 N LEU A 30 8.417 9.294 −4.422 1.00 16.34 N ATOM 189 CA LEU A 30 8.494 10.699 −4.743 1.00 15.26 C ATOM 190 CB LEU A 30 8.590 11.503 −3.448 1.00 15.20 C ATOM 191 CG LEU A 30 8.821 13.013 −3.506 1.00 14.70 C ATOM 192 CD1 LEU A 30 7.612 13.720 −4.106 1.00 13.91 C ATOM 193 CD2 LEU A 30 9.103 13.540 −2.110 1.00 15.07 C ATOM 194 C LEU A 30 9.768 10.865 −5.520 1.00 14.75 C ATOM 195 O LEU A 30 10.820 10.418 −5.079 1.00 15.24 O ATOM 196 N LYS A 31 9.681 11.515 −6.673 1.00 14.21 N ATOM 197 CA LYS A 31 10.836 11.703 −7.515 1.00 13.67 C ATOM 198 CB LYS A 31 10.774 10.760 −8.723 1.00 14.85 C ATOM 199 CG LYS A 31 11.932 10.899 −9.662 1.00 15.89 C ATOM 200 CD LYS A 31 11.819 9.944 −10.838 1.00 19.52 C ATOM 201 CE LYS A 31 12.921 10.178 −11.844 1.00 22.81 C ATOM 202 NZ LYS A 31 12.789 9.222 −12.987 1.00 27.06 N ATOM 203 C LYS A 31 10.854 13.156 −7.935 1.00 13.16 C ATOM 204 O LYS A 31 9.966 13.601 −8.658 1.00 13.34 O ATOM 205 N VAL A 32 11.860 13.882 −7.458 1.00 12.62 N ATOM 206 CA VAL A 32 11.998 15.309 −7.722 1.00 11.96 C ATOM 207 CB VAL A 32 12.264 16.078 −6.395 1.00 11.66 C ATOM 208 CG1 VAL A 32 12.475 17.570 −6.663 1.00 11.11 C ATOM 209 CG2 VAL A 32 11.137 15.844 −5.403 1.00 11.90 C ATOM 210 C VAL A 32 13.191 15.509 −8.658 1.00 12.56 C ATOM 211 O VAL A 32 14.326 15.142 −8.314 1.00 13.46 O ATOM 212 N THR A 33 12.956 16.076 −9.839 1.00 11.70 N ATOM 213 CA THR A 33 13.988 16.182 −10.858 1.00 12.55 C ATOM 214 CB THR A 33 13.748 15.161 −12.019 1.00 12.70 C ATOM 215 OG1 THR A 33 13.542 13.854 −11.471 1.00 13.48 O ATOM 216 CG2 THR A 33 14.918 15.097 −12.978 1.00 14.67 C ATOM 217 C THR A 33 14.116 17.599 −11.399 1.00 11.41 C ATOM 218 O THR A 33 13.142 18.205 −11.854 1.00 11.41 O ATOM 219 N TYR A 34 15.337 18.119 −11.343 1.00 11.75 N ATOM 220 CA TYR A 34 15.698 19.371 −11.959 1.00 12.44 C ATOM 221 CB TYR A 34 16.520 20.211 −10.995 1.00 12.68 C ATOM 222 CG TYR A 34 15.756 20.795 −9.820 1.00 10.94 C ATOM 223 CD1 TYR A 34 15.057 21.978 −9.959 1.00 11.31 C ATOM 224 CE1 TYR A 34 14.378 22.546 −8.880 1.00 9.98 C ATOM 225 CZ TYR A 34 14.432 21.943 −7.660 1.00 11.59 C ATOM 226 OH TYR A 34 13.787 22.548 −6.589 1.00 10.34 O ATOM 227 CE2 TYR A 34 15.138 20.768 −7.479 1.00 11.29 C ATOM 228 CD2 TYR A 34 15.806 20.193 −8.570 1.00 11.35 C ATOM 229 C TYR A 34 16.551 19.057 −13.178 1.00 13.57 C ATOM 230 O TYR A 34 17.664 18.553 −13.029 1.00 13.66 O ATOM 231 N GLY A 35 16.033 19.344 −14.374 1.00 13.84 N ATOM 232 CA GLY A 35 16.732 18.958 −15.616 1.00 15.18 C ATOM 233 C GLY A 35 16.800 17.440 −15.705 1.00 16.38 C ATOM 234 O GLY A 35 15.775 16.761 −15.785 1.00 16.96 O ATOM 235 N SER A 36 18.010 16.896 −15.662 1.00 17.77 N ATOM 236 CA SER A 36 18.150 15.438 −15.603 1.00 18.97 C ATOM 237 CB SER A 36 19.090 14.942 −16.696 1.00 19.64 C ATOM 238 OG SER A 36 20.385 15.464 −16.506 1.00 21.57 O ATOM 239 C SER A 36 18.637 14.949 −14.238 1.00 19.73 C ATOM 240 O SER A 36 18.903 13.764 −14.047 1.00 20.43 O ATOM 241 N LYS A 37 18.728 15.869 −13.281 1.00 19.10 N ATOM 242 CA LYS A 37 19.241 15.559 −11.950 1.00 20.22 C ATOM 243 CB LYS A 37 20.023 16.752 −11.382 1.00 19.94 C ATOM 244 CG LYS A 37 21.147 17.305 −12.241 1.00 23.65 C ATOM 245 CD LYS A 37 21.718 18.564 −11.584 1.00 23.68 C ATOM 246 CE LYS A 37 22.729 19.279 −12.471 1.00 28.46 C ATOM 247 NZ LYS A 37 23.991 18.497 −12.635 1.00 29.30 N ATOM 248 C LYS A 37 18.100 15.234 −10.999 1.00 19.27 C ATOM 249 O LYS A 37 17.367 16.135 −10.580 1.00 17.57 O ATOM 250 N THR A 38 17.958 13.963 −10.640 1.00 18.79 N ATOM 251 CA THR A 38 17.002 13.549 −9.619 1.00 18.53 C ATOM 252 CB THR A 38 16.510 12.088 −9.837 1.00 19.21 C ATOM 253 OG1 THR A 38 15.766 12.018 −11.064 1.00 19.55 O ATOM 254 CG2 THR A 38 15.620 11.605 −8.678 1.00 19.05 C ATOM 255 C THR A 38 17.589 13.750 −8.220 1.00 18.54 C ATOM 256 O THR A 38 18.716 13.357 −7.942 1.00 19.06 O ATOM 257 N VAL A 39 16.823 14.387 −7.342 1.00 16.92 N ATOM 258 CA VAL A 39 17.256 14.629 −5.964 1.00 17.12 C ATOM 259 CB VAL A 39 16.296 15.651 −5.283 1.00 16.75 C ATOM 260 CG1 VAL A 39 16.601 15.836 −3.789 1.00 17.52 C ATOM 261 CG2 VAL A 39 16.350 16.980 −6.017 1.00 15.54 C ATOM 262 C VAL A 39 17.315 13.338 −5.148 1.00 17.46 C ATOM 263 O VAL A 39 16.390 12.541 −5.164 1.00 17.06 O ATOM 264 N SER A 40 18.424 13.136 −4.435 1.00 17.68 N ATOM 265 CA SER A 40 18.441 12.193 −3.315 1.00 18.58 C ATOM 266 CB SER A 40 19.397 11.027 −3.552 1.00 19.00 C ATOM 267 OG SER A 40 20.692 11.505 −3.797 1.00 21.47 O ATOM 268 C SER A 40 18.843 12.987 −2.075 1.00 17.60 C ATOM 269 O SER A 40 19.448 14.046 −2.209 1.00 18.01 O ATOM 270 N ASN A 41 18.496 12.470 −0.901 1.00 18.90 N ATOM 271 CA ASN A 41 18.746 13.150 0.374 1.00 18.58 C ATOM 272 CB ASN A 41 18.370 12.236 1.538 1.00 18.85 C ATOM 273 CG ASN A 41 16.874 12.043 1.670 1.00 18.46 C ATOM 274 OD1 ASN A 41 16.381 10.933 1.825 1.00 21.16 O ATOM 275 ND2 ASN A 41 16.137 13.139 1.614 1.00 15.79 N ATOM 276 C ASN A 41 20.177 13.638 0.535 1.00 19.52 C ATOM 277 O ASN A 41 21.117 12.846 0.432 1.00 20.11 O ATOM 278 N GLY A 42 20.342 14.941 0.763 1.00 19.59 N ATOM 279 CA GLY A 42 21.663 15.520 1.022 1.00 20.73 C ATOM 280 C GLY A 42 22.414 15.963 −0.218 1.00 21.31 C ATOM 281 O GLY A 42 23.460 16.626 −0.121 1.00 22.32 O ATOM 282 N LEU A 43 21.887 15.603 −1.388 1.00 21.41 N ATOM 283 CA LEU A 43 22.442 16.031 −2.661 1.00 21.73 C ATOM 284 CB LEU A 43 21.517 15.623 −3.810 1.00 22.13 C ATOM 285 CG LEU A 43 21.984 15.984 −5.223 1.00 22.38 C ATOM 286 CD1 LEU A 43 23.238 15.189 −5.609 1.00 21.96 C ATOM 287 CD2 LEU A 43 20.871 15.734 −6.233 1.00 22.92 C ATOM 288 C LEU A 43 22.646 17.538 −2.690 1.00 21.61 C ATOM 289 O LEU A 43 21.733 18.301 −2.371 1.00 20.99 O ATOM 290 N GLU A 44 23.844 17.978 −3.044 1.00 21.67 N ATOM 291 CA GLU A 44 24.071 19.398 −3.182 1.00 21.58 C ATOM 292 CB GLU A 44 25.510 19.795 −2.838 1.00 22.31 C ATOM 293 CG GLU A 44 25.767 21.264 −3.143 1.00 23.85 C ATOM 294 CD GLU A 44 26.898 21.869 −2.338 1.00 26.59 C ATOM 295 OE1 GLU A 44 27.689 21.117 −1.717 1.00 28.58 O ATOM 296 OE2 GLU A 44 26.989 23.108 −2.334 1.00 27.92 O ATOM 297 C GLU A 44 23.709 19.854 −4.577 1.00 21.63 C ATOM 298 O GLU A 44 24.173 19.290 −5.574 1.00 21.73 O ATOM 299 N LEU A 45 22.856 20.871 −4.648 1.00 21.04 N ATOM 300 CA LEU A 45 22.528 21.507 −5.914 1.00 20.48 C ATOM 301 CB LEU A 45 21.049 21.313 −6.277 1.00 20.50 C ATOM 302 CG LEU A 45 20.593 19.903 −6.656 1.00 19.35 C ATOM 303 CD1 LEU A 45 19.071 19.851 −6.802 1.00 18.58 C ATOM 304 CD2 LEU A 45 21.269 19.409 −7.938 1.00 21.10 C ATOM 305 C LEU A 45 22.853 22.981 −5.857 1.00 20.71 C ATOM 306 O LEU A 45 22.694 23.618 −4.808 1.00 21.18 O ATOM 307 N LYS A 46 23.343 23.520 −6.970 1.00 20.32 N ATOM 308 CA LYS A 46 23.616 24.949 −7.055 1.00 20.19 C ATOM 309 CB LYS A 46 24.571 25.284 −8.209 1.00 21.02 C ATOM 310 CG LYS A 46 25.895 24.535 −8.156 1.00 23.71 C ATOM 311 CD LYS A 46 26.979 25.263 −8.955 1.00 28.47 C ATOM 312 CE LYS A 46 26.486 25.721 −10.324 1.00 30.79 C ATOM 313 NZ LYS A 46 27.442 26.682 −10.955 1.00 32.21 N ATOM 314 C LYS A 46 22.309 25.705 −7.222 1.00 19.43 C ATOM 315 O LYS A 46 21.385 25.207 −7.870 1.00 18.78 O ATOM 316 N PRO A 47 22.219 26.915 −6.648 1.00 19.03 N ATOM 317 CA PRO A 47 21.036 27.749 −6.838 1.00 19.24 C ATOM 318 CB PRO A 47 21.468 29.098 −6.242 1.00 19.42 C ATOM 319 CG PRO A 47 22.429 28.721 −5.192 1.00 19.39 C ATOM 320 CD PRO A 47 23.201 27.563 −5.759 1.00 19.56 C ATOM 321 C PRO A 47 20.602 27.882 −8.311 1.00 18.88 C ATOM 322 O PRO A 47 19.403 27.843 −8.608 1.00 18.80 O ATOM 323 N SER A 48 21.553 28.041 −9.235 1.00 19.58 N ATOM 324 CA SER A 48 21.211 28.192 −10.651 1.00 19.51 C ATOM 325 CB SER A 48 22.468 28.474 −11.486 1.00 20.07 C ATOM 326 OG SER A 48 23.320 27.347 −11.455 1.00 22.63 O ATOM 327 C SER A 48 20.473 26.977 −11.226 1.00 19.17 C ATOM 328 O SER A 48 19.767 27.096 −12.216 1.00 19.62 O ATOM 329 N MET A 49 20.653 25.812 −10.617 1.00 18.45 N ATOM 330 CA MET A 49 19.988 24.603 −11.080 1.00 18.55 C ATOM 331 CB MET A 49 20.745 23.363 −10.589 1.00 18.68 C ATOM 332 CG MET A 49 22.172 23.209 −11.148 1.00 20.25 C ATOM 333 SD MET A 49 23.138 22.099 −10.126 1.00 23.81 S ATOM 334 CE MET A 49 24.770 22.202 −10.862 1.00 22.41 C ATOM 335 C MET A 49 18.523 24.533 −10.645 1.00 17.01 C ATOM 336 O MET A 49 17.742 23.780 −11.216 1.00 16.05 O ATOM 337 N VAL A 50 18.156 25.331 −9.644 1.00 14.99 N ATOM 338 CA VAL A 50 16.839 25.205 −8.993 1.00 13.94 C ATOM 339 CB VAL A 50 16.974 24.654 −7.542 1.00 12.87 C ATOM 340 CG1 VAL A 50 17.667 23.304 −7.565 1.00 13.50 C ATOM 341 CG2 VAL A 50 17.724 25.622 −6.631 1.00 14.15 C ATOM 342 C VAL A 50 15.977 26.468 −9.071 1.00 12.86 C ATOM 343 O VAL A 50 15.020 26.634 −8.300 1.00 12.24 O ATOM 344 N THR A 51 16.289 27.345 −10.018 1.00 13.48 N ATOM 345 CA THR A 51 15.551 28.596 −10.204 1.00 13.59 C ATOM 346 CB THR A 51 16.220 29.446 −11.302 1.00 14.31 C ATOM 347 OG1 THR A 51 16.404 28.628 −12.463 1.00 16.31 O ATOM 348 CG2 THR A 51 17.574 29.940 −10.845 1.00 14.50 C ATOM 349 C THR A 51 14.060 28.386 −10.544 1.00 13.98 C ATOM 350 O THR A 51 13.204 29.184 −10.159 1.00 14.64 O ATOM 351 N HIS A 52 13.754 27.313 −11.276 1.00 13.33 N ATOM 352 CA HIS A 52 12.386 26.982 −11.647 1.00 13.04 C ATOM 353 CB HIS A 52 12.282 26.832 −13.151 1.00 13.68 C ATOM 354 CG HIS A 52 12.477 28.114 −13.884 1.00 14.46 C ATOM 355 ND1 HIS A 52 13.708 28.721 −14.014 1.00 16.14 N ATOM 356 CE1 HIS A 52 13.565 29.843 −14.699 1.00 16.08 C ATOM 357 NE2 HIS A 52 12.291 29.983 −15.011 1.00 18.18 N ATOM 358 CD2 HIS A 52 11.588 28.920 −14.500 1.00 15.40 C ATOM 359 C HIS A 52 11.971 25.678 −10.991 1.00 11.09 C ATOM 360 O HIS A 52 12.821 24.887 −10.577 1.00 11.88 O ATOM 361 N GLN A 53 10.662 25.453 −10.916 1.00 11.62 N ATOM 362 CA GLN A 53 10.182 24.293 −10.182 1.00 11.19 C ATOM 363 CB GLN A 53 8.651 24.285 −10.044 1.00 10.34 C ATOM 364 CG GLN A 53 7.900 24.105 −11.336 1.00 11.51 C ATOM 365 CD GLN A 53 6.494 24.537 −11.186 1.00 15.07 C ATOM 366 OE1 GLN A 53 6.220 25.739 −11.055 1.00 15.59 O ATOM 367 NE2 GLN A 53 5.583 23.584 −11.193 1.00 14.39 N ATOM 368 C GLN A 53 10.667 22.995 −10.820 1.00 10.53 C ATOM 369 O GLN A 53 10.759 22.893 −12.064 1.00 11.42 O ATOM 370 N PRO A 54 10.977 21.992 −9.983 1.00 10.57 N ATOM 371 CA PRO A 54 11.353 20.695 −10.519 1.00 10.47 C ATOM 372 CB PRO A 54 11.933 19.974 −9.312 1.00 10.85 C ATOM 373 CG PRO A 54 11.188 20.546 −8.128 1.00 10.74 C ATOM 374 CD PRO A 54 10.947 22.001 −8.499 1.00 9.97 C ATOM 375 C PRO A 54 10.128 19.932 −10.989 1.00 11.10 C ATOM 376 O PRO A 54 8.977 20.289 −10.664 1.00 11.18 O ATOM 377 N ARG A 55 10.362 18.893 −11.779 1.00 11.44 N ATOM 378 CA ARG A 55 9.305 17.929 −12.042 1.00 12.31 C ATOM 379 CB ARG A 55 9.648 17.104 −13.267 1.00 13.37 C ATOM 380 CG ARG A 55 9.553 17.863 −14.555 1.00 16.91 C ATOM 381 CD ARG A 55 9.591 16.900 −15.732 1.00 19.52 C ATOM 382 NE ARG A 55 10.788 16.061 −15.678 1.00 21.39 N ATOM 383 CZ ARG A 55 12.021 16.508 −15.883 1.00 20.16 C ATOM 384 NH1 ARG A 55 12.238 17.792 −16.157 1.00 22.79 N ATOM 385 NH2 ARG A 55 13.049 15.669 −15.806 1.00 21.29 N ATOM 386 C ARG A 55 9.191 17.027 −10.841 1.00 12.50 C ATOM 387 O ARG A 55 10.201 16.551 −10.328 1.00 12.92 O ATOM 388 N VAL A 56 7.978 16.792 −10.366 1.00 11.65 N ATOM 389 CA VAL A 56 7.798 15.967 −9.178 1.00 11.93 C ATOM 390 CB VAL A 56 7.371 16.797 −7.952 1.00 12.02 C ATOM 391 CG1 VAL A 56 7.209 15.940 −6.723 1.00 12.50 C ATOM 392 CG2 VAL A 56 8.393 17.899 −7.678 1.00 11.26 C ATOM 393 C VAL A 56 6.797 14.888 −9.487 1.00 13.55 C ATOM 394 O VAL A 56 5.595 15.142 −9.510 1.00 13.06 O ATOM 395 N GLU A 57 7.319 13.690 −9.734 1.00 13.98 N ATOM 396 CA GLU A 57 6.489 12.547 −10.035 1.00 15.76 C ATOM 397 CB GLU A 57 7.231 11.563 −10.946 1.00 15.61 C ATOM 398 CG GLU A 57 7.449 12.137 −12.326 1.00 16.98 C ATOM 399 CD GLU A 57 8.192 11.204 −13.291 1.00 19.08 C ATOM 400 OE1 GLU A 57 8.359 9.998 −12.990 1.00 24.85 O ATOM 401 OE2 GLU A 57 8.589 11.703 −14.370 1.00 25.69 O ATOM 402 C GLU A 57 6.079 11.914 −8.737 1.00 15.53 C ATOM 403 O GLU A 57 6.872 11.803 −7.795 1.00 15.86 O ATOM 404 N VAL A 58 4.804 11.548 −8.666 1.00 15.80 N ATOM 405 CA VAL A 58 4.287 10.872 −7.488 1.00 17.11 C ATOM 406 CB VAL A 58 3.144 11.638 −6.767 1.00 17.67 C ATOM 407 CG1 VAL A 58 3.577 13.081 −6.451 1.00 16.49 C ATOM 408 CG2 VAL A 58 1.843 11.613 −7.563 1.00 19.47 C ATOM 409 C VAL A 58 3.846 9.466 −7.863 1.00 17.01 C ATOM 410 O VAL A 58 3.220 9.241 −8.923 1.00 17.90 O ATOM 411 N GLY A 59 4.182 8.529 −6.994 1.00 16.97 N ATOM 412 CA GLY A 59 3.787 7.144 −7.179 1.00 17.19 C ATOM 413 C GLY A 59 2.376 6.870 −6.695 1.00 17.33 C ATOM 414 O GLY A 59 1.438 7.647 −6.932 1.00 16.56 O ATOM 415 N GLY A 60 2.224 5.744 −6.008 1.00 18.31 N ATOM 416 CA GLY A 60 0.909 5.284 −5.586 1.00 19.22 C ATOM 417 C GLY A 60 0.259 4.294 −6.557 1.00 20.17 C ATOM 418 O GLY A 60 0.885 3.886 −7.546 1.00 20.71 O ATOM 419 N ASN A 61 −0.993 3.931 −6.264 1.00 20.95 N ATOM 420 CA ASN A 61 −1.725 2.864 −6.968 1.00 22.07 C ATOM 421 CB ASN A 61 −2.785 2.245 −6.045 1.00 22.28 C ATOM 422 CG ASN A 61 −2.244 1.112 −5.205 1.00 25.63 C ATOM 423 OD1 ASN A 61 −1.524 0.248 −5.695 1.00 28.95 O ATOM 424 ND2 ASN A 61 −2.612 1.096 −3.935 1.00 28.97 N ATOM 425 C ASN A 61 −2.435 3.335 −8.218 1.00 21.66 C ATOM 426 O ASN A 61 −2.480 2.617 −9.221 1.00 22.35 O ATOM 427 N ASP A 62 −3.044 4.515 −8.137 1.00 21.12 N ATOM 428 CA ASP A 62 −3.845 5.041 −9.237 1.00 21.14 C ATOM 429 CB ASP A 62 −5.113 4.184 −9.483 1.00 21.61 C ATOM 430 CG ASP A 62 −6.047 4.134 −8.274 1.00 22.61 C ATOM 431 OD1 ASP A 62 −6.580 5.188 −7.876 1.00 24.91 O ATOM 432 OD2 ASP A 62 −6.287 3.023 −7.746 1.00 24.60 O ATOM 433 C ASP A 62 −4.198 6.506 −9.049 1.00 20.60 C ATOM 434 O ASP A 62 −3.890 7.115 −8.023 1.00 19.85 O ATOM 435 N MET A 63 −4.862 7.052 −10.061 1.00 19.74 N ATOM 436 CA MET A 63 −5.155 8.482 −10.132 1.00 19.71 C ATOM 437 CB MET A 63 −5.462 8.876 −11.578 1.00 19.87 C ATOM 438 CG MET A 63 −4.252 8.824 −12.474 1.00 19.72 C ATOM 439 SD MET A 63 −4.641 9.187 −14.196 1.00 23.34 S ATOM 440 CE MET A 63 −5.239 10.851 −14.073 1.00 23.23 C ATOM 441 C MET A 63 −6.279 8.949 −9.219 1.00 18.85 C ATOM 442 O MET A 63 −6.583 10.142 −9.183 1.00 18.07 O ATOM 443 N ARG A 64 −6.908 8.023 −8.496 1.00 18.97 N ATOM 444 CA ARG A 64 −8.016 8.387 −7.616 1.00 18.87 C ATOM 445 CB ARG A 64 −9.180 7.387 −7.736 1.00 19.18 C ATOM 446 CG ARG A 64 −9.836 7.376 −9.104 1.00 21.09 C ATOM 447 CD ARG A 64 −11.063 6.463 −9.113 1.00 21.95 C ATOM 448 NE ARG A 64 −12.268 7.086 −8.552 1.00 26.78 N ATOM 449 CZ ARG A 64 −12.827 6.756 −7.387 1.00 28.44 C ATOM 450 NH1 ARG A 64 −12.300 5.797 −6.628 1.00 29.72 N ATOM 451 NH2 ARG A 64 −13.930 7.383 −6.977 1.00 30.04 N ATOM 452 C ARG A 64 −7.566 8.560 −6.164 1.00 18.10 C ATOM 453 O ARG A 64 −8.379 8.828 −5.282 1.00 17.60 O ATOM 454 N THR A 65 −6.265 8.402 −5.928 1.00 16.62 N ATOM 455 CA THR A 65 −5.654 8.824 −4.671 1.00 16.22 C ATOM 456 CB THR A 65 −4.607 7.817 −4.152 1.00 16.94 C ATOM 457 OG1 THR A 65 −5.246 6.567 −3.857 1.00 19.49 O ATOM 458 CG2 THR A 65 −3.955 8.323 −2.872 1.00 18.53 C ATOM 459 C THR A 65 −4.985 10.164 −4.948 1.00 14.81 C ATOM 460 O THR A 65 −4.329 10.331 −5.994 1.00 15.24 O ATOM 461 N PHE A 66 −5.157 11.106 −4.021 1.00 13.11 N ATOM 462 CA PHE A 66 −4.630 12.459 −4.197 1.00 12.59 C ATOM 463 CB PHE A 66 −5.768 13.486 −4.208 1.00 13.15 C ATOM 464 CG PHE A 66 −6.740 13.272 −5.318 1.00 14.50 C ATOM 465 CD1 PHE A 66 −6.507 13.820 −6.573 1.00 14.94 C ATOM 466 CE1 PHE A 66 −7.398 13.585 −7.635 1.00 17.78 C ATOM 467 CZ PHE A 66 −8.527 12.785 −7.424 1.00 17.85 C ATOM 468 CE2 PHE A 66 −8.760 12.220 −6.174 1.00 18.68 C ATOM 469 CD2 PHE A 66 −7.855 12.454 −5.126 1.00 16.22 C ATOM 470 C PHE A 66 −3.599 12.811 −3.143 1.00 12.46 C ATOM 471 O PHE A 66 −3.707 12.405 −1.987 1.00 12.11 O ATOM 472 N TYR A 67 −2.626 13.621 −3.564 1.00 11.47 N ATOM 473 CA TYR A 67 −1.486 13.976 −2.740 1.00 10.69 C ATOM 474 CB TYR A 67 −0.231 13.287 −3.283 1.00 11.54 C ATOM 475 CG TYR A 67 −0.299 11.783 −3.258 1.00 12.17 C ATOM 476 CD1 TYR A 67 −0.789 11.084 −4.356 1.00 12.88 C ATOM 477 CE1 TYR A 67 −0.863 9.696 −4.344 1.00 15.49 C ATOM 478 CZ TYR A 67 −0.467 9.007 −3.229 1.00 14.40 C ATOM 479 OH TYR A 67 −0.544 7.623 −3.252 1.00 16.14 O ATOM 480 CE2 TYR A 67 0.030 9.669 −2.108 1.00 13.73 C ATOM 481 CD2 TYR A 67 0.096 11.058 −2.127 1.00 12.45 C ATOM 482 C TYR A 67 −1.259 15.485 −2.645 1.00 10.61 C ATOM 483 O TYR A 67 −1.557 16.247 −3.578 1.00 10.82 O ATOM 484 N THR A 68 −0.704 15.890 −1.505 1.00 10.52 N ATOM 485 CA THR A 68 −0.263 17.277 −1.282 1.00 10.36 C ATOM 486 CB THR A 68 −0.908 17.865 −0.022 1.00 10.43 C ATOM 487 OG1 THR A 68 −2.267 18.195 −0.329 1.00 10.77 O ATOM 488 CG2 THR A 68 −0.187 19.143 0.444 1.00 11.32 C ATOM 489 C THR A 68 1.253 17.302 −1.166 1.00 9.84 C ATOM 490 O THR A 68 1.842 16.490 −0.463 1.00 10.24 O ATOM 491 N LEU A 69 1.865 18.253 −1.877 1.00 9.28 N ATOM 492 CA LEU A 69 3.304 18.502 −1.870 1.00 8.99 C ATOM 493 CB LEU A 69 3.862 18.468 −3.294 1.00 9.04 C ATOM 494 CG LEU A 69 5.358 18.743 −3.419 1.00 10.30 C ATOM 495 CD1 LEU A 69 6.159 17.556 −2.945 1.00 11.75 C ATOM 496 CD2 LEU A 69 5.683 19.017 −4.870 1.00 10.37 C ATOM 497 C LEU A 69 3.605 19.862 −1.244 1.00 8.91 C ATOM 498 O LEU A 69 3.053 20.894 −1.650 1.00 9.05 O ATOM 499 N VAL A 70 4.492 19.828 −0.254 1.00 8.68 N ATOM 500 CA VAL A 70 4.982 20.994 0.472 1.00 8.66 C ATOM 501 CB VAL A 70 4.811 20.786 1.982 1.00 8.76 C ATOM 502 CG1 VAL A 70 5.428 21.967 2.765 1.00 11.73 C ATOM 503 CG2 VAL A 70 3.345 20.583 2.320 1.00 11.81 C ATOM 504 C VAL A 70 6.464 21.117 0.207 1.00 7.88 C ATOM 505 O VAL A 70 7.188 20.135 0.314 1.00 8.44 O ATOM 506 N MET A 71 6.935 22.326 −0.079 1.00 8.16 N ATOM 507 CA MET A 71 8.375 22.575 −0.162 1.00 7.63 C ATOM 508 CB MET A 71 8.801 22.859 −1.605 1.00 7.87 C ATOM 509 CG MET A 71 10.298 23.096 −1.763 1.00 8.73 C ATOM 510 SD MET A 71 10.624 23.912 −3.339 1.00 8.90 S ATOM 511 CE MET A 71 12.412 24.075 −3.272 1.00 10.72 C ATOM 512 C MET A 71 8.737 23.719 0.761 1.00 6.96 C ATOM 513 O MET A 71 8.220 24.833 0.606 1.00 7.60 O ATOM 514 N VAL A 72 9.636 23.441 1.714 1.00 7.01 N ATOM 515 CA VAL A 72 10.044 24.447 2.711 1.00 7.91 C ATOM 516 CB VAL A 72 9.396 24.178 4.105 1.00 7.93 C ATOM 517 CG1 VAL A 72 7.883 24.346 4.040 1.00 10.28 C ATOM 518 CG2 VAL A 72 9.742 22.769 4.625 1.00 9.84 C ATOM 519 C VAL A 72 11.557 24.552 2.884 1.00 8.26 C ATOM 520 O VAL A 72 12.276 23.586 2.628 1.00 8.13 O ATOM 521 N ASP A 73 12.011 25.743 3.298 1.00 8.24 N ATOM 522 CA ASP A 73 13.401 25.974 3.737 1.00 8.48 C ATOM 523 CB ASP A 73 14.015 27.175 3.029 1.00 9.04 C ATOM 524 CG ASP A 73 15.486 27.391 3.379 1.00 10.14 C ATOM 525 OD1 ASP A 73 16.011 26.721 4.280 1.00 9.98 O ATOM 526 OD2 ASP A 73 16.088 28.275 2.747 1.00 12.13 O ATOM 527 C ASP A 73 13.346 26.211 5.247 1.00 8.65 C ATOM 528 O ASP A 73 12.906 27.252 5.703 1.00 9.12 O ATOM 529 N PRO A 74 13.782 25.226 6.033 1.00 9.39 N ATOM 530 CA PRO A 74 13.805 25.409 7.489 1.00 9.78 C ATOM 531 CB PRO A 74 13.891 23.978 7.999 1.00 10.36 C ATOM 532 CG PRO A 74 14.702 23.280 6.961 1.00 9.48 C ATOM 533 CD PRO A 74 14.228 23.869 5.642 1.00 9.67 C ATOM 534 C PRO A 74 15.026 26.203 7.961 1.00 10.88 C ATOM 535 O PRO A 74 15.128 26.513 9.155 1.00 11.53 O ATOM 536 N ASP A 75 15.911 26.550 7.024 1.00 11.03 N ATOM 537 CA ASP A 75 17.165 27.260 7.311 1.00 11.09 C ATOM 538 CB ASP A 75 18.338 26.511 6.659 1.00 11.39 C ATOM 539 CG ASP A 75 18.491 25.119 7.195 1.00 11.34 C ATOM 540 OD1 ASP A 75 18.436 24.972 8.420 1.00 13.96 O ATOM 541 OD2 ASP A 75 18.635 24.129 6.430 1.00 13.93 O ATOM 542 C ASP A 75 17.140 28.703 6.830 1.00 11.19 C ATOM 543 O ASP A 75 18.185 29.242 6.482 1.00 12.38 O ATOM 544 N ALA A 76 15.955 29.316 6.793 1.00 11.30 N ATOM 545 CA ALA A 76 15.777 30.669 6.274 1.00 12.10 C ATOM 546 CB ALA A 76 14.511 30.740 5.405 1.00 12.47 C ATOM 547 C ALA A 76 15.685 31.690 7.412 1.00 12.33 C ATOM 548 O ALA A 76 14.973 31.464 8.389 1.00 12.27 O ATOM 549 N PRO A 77 16.405 32.821 7.300 1.00 13.41 N ATOM 550 CA PRO A 77 17.378 33.201 6.264 1.00 13.79 C ATOM 551 CB PRO A 77 17.556 34.718 6.500 1.00 14.09 C ATOM 552 CG PRO A 77 16.417 35.130 7.423 1.00 15.20 C ATOM 553 CD PRO A 77 16.202 33.910 8.271 1.00 13.53 C ATOM 554 C PRO A 77 18.739 32.480 6.323 1.00 14.46 C ATOM 555 O PRO A 77 19.449 32.445 5.318 1.00 14.37 O ATOM 556 N SER A 78 19.100 31.917 7.474 1.00 14.30 N ATOM 557 CA SER A 78 20.381 31.226 7.646 1.00 15.55 C ATOM 558 CB SEA A 78 21.394 32.166 8.307 1.00 16.01 C ATOM 559 OG SEA A 78 22.591 31.496 8.666 1.00 20.32 O ATOM 560 C SEA A 78 20.214 30.012 8.532 1.00 14.81 C ATOM 561 O SEA A 78 19.428 30.050 9.477 1.00 13.88 O ATOM 562 N PRO A 79 20.981 28.931 8.277 1.00 15.25 N ATOM 563 CA PRO A 79 20.892 27.817 9.219 1.00 15.73 C ATOM 564 CB PRO A 79 21.821 26.753 8.620 1.00 15.83 C ATOM 565 CG PRO A 79 22.718 27.490 7.683 1.00 15.98 C ATOM 566 CD PRO A 79 21.911 28.648 7.166 1.00 15.07 C ATOM 567 C PRO A 79 21.312 28.196 10.646 1.00 15.88 C ATOM 568 O PRO A 79 20.826 27.608 11.601 1.00 16.58 O ATOM 569 N SER A 80 22.175 29.197 10.791 1.00 16.07 N ATOM 570 CA SER A 80 22.585 29.631 12.140 1.00 16.19 C ATOM 571 CB SER A 80 24.035 30.105 12.116 1.00 17.04 C ATOM 572 OG SEA A 80 24.132 31.288 11.370 1.00 17.95 O ATOM 573 C SEA A 80 21.676 30.713 12.746 1.00 16.20 C ATOM 574 O SER A 80 21.848 31.107 13.900 1.00 15.71 O ATOM 575 N ASP A 81 20.691 31.170 11.978 1.00 15.78 N ATOM 576 CA ASP A 81 19.703 32.137 12.445 1.00 15.53 C ATOM 577 CB ASP A 81 20.245 33.562 12.278 1.00 17.12 C ATOM 578 CG ASP A 81 19.265 34.646 12.725 1.00 19.91 C ATOM 579 OD1 ASP A 81 18.211 34.363 13.338 1.00 20.54 O ATOM 580 OD2 ASP A 81 19.575 35.820 12.444 1.00 26.03 O ATOM 581 C ASP A 81 18.409 31.911 11.633 1.00 14.33 C ATOM 582 O ASP A 81 18.048 32.710 10.775 1.00 14.61 O ATOM 583 N PRO A 82 17.725 30.791 11.893 1.00 13.32 N ATOM 584 CA PRO A 82 16.580 30.414 11.055 1.00 12.43 C ATOM 585 CB PRO A 82 16.513 28.895 11.247 1.00 12.84 C ATOM 586 CG PRO A 82 16.989 28.687 12.670 1.00 12.18 C ATOM 587 CD PRO A 82 18.021 29.771 12.922 1.00 13.84 C ATOM 588 C PRO A 82 15.273 31.072 11.490 1.00 12.14 C ATOM 589 O PRO A 82 14.271 30.393 11.728 1.00 11.56 O ATOM 590 N ASN A 83 15.272 32.401 11.571 1.00 11.82 N ATOM 591 CA ASN A 83 14.128 33.108 12.156 1.00 12.00 C ATOM 592 CB ASN A 83 14.508 34.544 12.560 1.00 12.64 C ATOM 593 CG ASN A 83 14.971 35.375 11.398 1.00 13.45 C ATOM 594 OD1 ASN A 83 14.177 35.749 10.530 1.00 15.06 O ATOM 595 ND2 ASN A 83 16.273 35.686 11.365 1.00 14.12 N ATOM 596 C ASN A 83 12.844 33.101 11.322 1.00 12.24 C ATOM 597 O ASN A 83 11.772 33.445 11.831 1.00 12.21 O ATOM 598 N LED A 84 12.963 32.688 10.062 1.00 11.14 N ATOM 599 CA LED A 84 11.785 32.538 9.199 1.00 10.21 C ATOM 600 CB LEU A 84 12.025 33.158 7.823 1.00 11.50 C ATOM 601 CG LEU A 84 12.373 34.657 7.770 1.00 11.33 C ATOM 602 CD1 LEU A 84 12.589 35.063 6.327 1.00 15.59 C ATOM 603 CD2 LEU A 84 11.297 35.497 8.466 1.00 12.96 C ATOM 604 C LEU A 84 11.344 31.083 9.053 1.00 10.32 C ATOM 605 O LEU A 84 10.454 30.792 8.237 1.00 9.88 O ATOM 606 N ARG A 85 11.952 30.177 9.815 1.00 10.23 N ATOM 607 CA ARG A 85 11.593 28.761 9.745 1.00 9.62 C ATOM 608 CB ARG A 85 12.312 27.963 10.847 1.00 10.21 C ATOM 609 CG ARG A 85 11.844 26.508 10.923 1.00 10.25 C ATOM 610 CD ARG A 85 12.644 25.729 11.960 1.00 12.94 C ATOM 611 NE ARG A 85 14.029 25.577 11.550 1.00 12.84 N ATOM 612 CZ ARG A 85 14.949 24.921 12.249 1.00 16.33 C ATOM 613 NH1 ARG A 85 14.632 24.365 13.420 1.00 18.08 N ATOM 614 NH2 ARG A 85 16.184 24.836 11.765 1.00 16.17 N ATOM 615 C ARG A 85 10.107 28.562 9.971 1.00 8.80 C ATOM 616 O ARG A 85 9.591 29.077 10.949 1.00 10.26 O ATOM 617 N GLU A 86 9.409 27.792 9.129 1.00 8.68 N ATOM 618 CA GLU A 86 9.852 27.289 7.822 1.00 8.00 C ATOM 619 CB GLU A 86 9.146 25.954 7.521 1.00 8.19 C ATOM 620 CG GLU A 86 9.428 24.861 8.559 1.00 9.10 C ATOM 621 CD GLU A 86 8.342 23.794 8.674 1.00 9.77 C ATOM 622 OE1 GLU A 86 7.510 23.643 7.762 1.00 11.38 O ATOM 623 OE2 GLU A 86 8.320 23.104 9.725 1.00 12.78 O ATOM 624 C GLU A 86 9.437 28.274 6.742 1.00 7.90 C ATOM 625 O GLU A 86 8.319 28.766 6.786 1.00 8.50 O ATOM 626 N TYR A 87 10.292 28.539 5.754 1.00 7.01 N ATOM 627 CA TYR A 87 9.869 29.427 4.672 1.00 7.34 C ATOM 628 CB TYR A 87 11.011 30.335 4.156 1.00 7.65 C ATOM 629 CG TYR A 87 10.515 31.364 3.174 1.00 7.73 C ATOM 630 CD1 TYR A 87 10.061 32.602 3.624 1.00 8.89 C ATOM 631 CE1 TYR A 87 9.543 33.548 2.727 1.00 9.48 C ATOM 632 CZ TYR A 87 9.525 33.251 1.370 1.00 9.35 C ATOM 633 OH TYR A 87 9.047 34.170 0.475 1.00 11.45 O ATOM 634 CE2 TYR A 87 9.992 32.012 0.910 1.00 8.08 C ATOM 635 CD2 TYR A 87 10.469 31.085 1.808 1.00 8.96 C ATOM 636 C TYR A 87 9.237 28.585 3.556 1.00 6.85 C ATOM 637 O TYR A 87 9.871 27.679 3.014 1.00 7.21 O ATOM 638 N LEU A 88 7.989 28.873 3.222 1.00 7.27 N ATOM 639 CA LEU A 88 7.232 28.062 2.268 1.00 7.42 C ATOM 640 CB LEU A 88 5.731 28.206 2.569 1.00 7.51 C ATOM 641 CG LEU A 88 4.811 27.408 1.644 1.00 6.76 C ATOM 642 CD1 LEU A 88 5.041 25.889 1.840 1.00 8.93 C ATOM 643 CD2 LEU A 88 3.353 27.778 1.883 1.00 8.72 C ATOM 644 C LEU A 88 7.555 28.458 0.832 1.00 7.42 C ATOM 645 O LEU A 88 7.187 29.557 0.384 1.00 7.91 O ATOM 646 N HIS A 89 8.248 27.566 0.117 1.00 6.48 N ATOM 647 CA HIS A 89 8.633 27.804 −1.276 1.00 7.32 C ATOM 648 CB HIS A 89 9.973 27.151 −1.594 1.00 7.14 C ATOM 649 CG HIS A 89 11.147 27.951 −1.117 1.00 7.90 C ATOM 650 ND1 HIS A 89 11.797 28.857 −1.926 1.00 9.32 N ATOM 651 CE1 HIS A 89 12.759 29.446 −1.231 1.00 9.87 C ATOM 652 NE2 HIS A 89 12.744 28.968 −0.003 1.00 9.07 N ATOM 653 CD2 HIS A 89 11.732 28.038 0.102 1.00 9.94 C ATOM 654 C HIS A 89 7.615 27.386 −2.328 1.00 7.51 C ATOM 655 O HIS A 89 7.616 27.948 −3.421 1.00 9.17 O ATOM 656 N TRP A 90 6.740 26.429 −2.007 1.00 8.06 N ATOM 657 CA TRP A 90 5.845 25.845 −3.012 1.00 7.76 C ATOM 658 CB TRP A 90 6.687 24.975 −3.954 1.00 7.32 C ATOM 659 CG TRP A 90 6.069 24.527 −5.241 1.00 8.01 C ATOM 660 CD1 TRP A 90 5.055 25.112 −5.967 1.00 8.69 C ATOM 661 NE1 TRP A 90 4.816 24.372 −7.113 1.00 9.57 N ATOM 662 CE2 TRP A 90 5.704 23.322 −7.155 1.00 8.74 C ATOM 663 CD2 TRP A 90 6.507 23.396 −6.000 1.00 7.96 C ATOM 664 CE3 TRP A 90 7.505 22.419 −5.804 1.00 7.84 C ATOM 665 CZ3 TRP A 90 7.661 21.426 −6.760 1.00 8.94 C ATOM 666 CH2 TRP A 90 6.847 21.381 −7.899 1.00 9.47 C ATOM 667 CZ2 TRP A 90 5.864 22.311 −8.113 1.00 8.43 C ATOM 668 C TRP A 90 4.819 24.995 −2.299 1.00 7.64 C ATOM 669 O TRP A 90 5.151 24.325 −1.319 1.00 7.71 O ATOM 670 N LEU A 91 3.585 25.000 −2.800 1.00 8.34 N ATOM 671 CA LEU A 91 2.521 24.186 −2.228 1.00 8.60 C ATOM 672 CB LEU A 91 1.800 24.991 −1.160 1.00 8.11 C ATOM 673 CG LEU A 91 0.763 24.226 −0.324 1.00 9.82 C ATOM 674 CD1 LEU A 91 1.370 23.023 0.417 1.00 12.77 C ATOM 675 CD2 LEU A 91 0.066 25.188 0.630 1.00 12.11 C ATOM 676 C LEU A 91 1.562 23.779 −3.342 1.00 9.49 C ATOM 677 O LEU A 91 1.033 24.646 −4.035 1.00 9.62 O ATOM 678 N VAL A 92 1.359 22.470 −3.501 1.00 9.41 N ATOM 679 CA VAL A 92 0.487 21.941 −4.538 1.00 10.23 C ATOM 680 CB VAL A 92 1.298 21.302 −5.677 1.00 9.84 C ATOM 681 CG1 VAL A 92 0.403 20.846 −6.831 1.00 10.65 C ATOM 682 CG2 VAL A 92 2.385 22.268 −6.209 1.00 10.59 C ATOM 683 C VAL A 92 −0.385 20.864 −3.907 1.00 10.75 C ATOM 684 O VAL A 92 0.100 20.023 −3.132 1.00 11.18 O ATOM 685 N THR A 93 −1.667 20.857 −4.251 1.00 10.91 N ATOM 686 CA THR A 93 −2.595 19.882 −3.655 1.00 11.96 C ATOM 687 CB THR A 93 −3.603 20.559 −2.727 1.00 12.12 C ATOM 688 OG1 THR A 93 −4.412 21.493 −3.482 1.00 14.35 O ATOM 689 CG2 THR A 93 −2.862 21.282 −1.592 1.00 14.25 C ATOM 690 C THR A 93 −3.338 19.117 −4.742 1.00 11.26 C ATOM 691 O THR A 93 −3.227 19.450 −5.904 1.00 11.58 O ATOM 692 N ASP A 94 −4.101 18.099 −4.338 1.00 11.43 N ATOM 693 CA ASP A 94 −4.919 17.313 −5.280 1.00 11.92 C ATOM 694 CB ASP A 94 −6.093 18.151 −5.837 1.00 12.03 C ATOM 695 CG ASP A 94 −7.249 18.318 −4.857 1.00 13.96 C ATOM 696 OD1 ASP A 94 −7.226 17.802 −3.717 1.00 12.99 O ATOM 697 OD2 ASP A 94 −8.202 19.019 −5.264 1.00 19.42 O ATOM 698 C ASP A 94 −4.078 16.789 −6.442 1.00 11.54 C ATOM 699 O ASP A 94 −4.545 16.758 −7.599 1.00 12.67 O ATOM 700 N ILE A 95 −2.847 16.350 −6.158 1.00 11.13 N ATOM 701 CA ILE A 95 −2.035 15.704 −7.184 1.00 10.88 C ATOM 702 CB ILE A 95 −0.543 15.699 −6.806 1.00 10.33 C ATOM 703 CG1 ILE A 95 −0.030 17.136 −6.564 1.00 10.03 C ATOM 704 CD1 ILE A 95 1.299 17.205 −5.814 1.00 10.35 C ATOM 705 CG2 ILE A 95 0.257 14.960 −7.877 1.00 11.90 C ATOM 706 C ILE A 95 −2.483 14.249 −7.369 1.00 10.78 C ATOM 707 O ILE A 95 −2.428 13.475 −6.415 1.00 11.23 O ATOM 708 N PRO A 96 −2.936 13.878 −8.586 1.00 11.11 N ATOM 709 CA PRO A 96 −3.361 12.480 −8.740 1.00 11.36 C ATOM 710 CB PRO A 96 −3.968 12.456 −10.147 1.00 11.86 C ATOM 711 CG PRO A 96 −4.285 13.873 −10.467 1.00 11.17 C ATOM 712 CD PRO A 96 −3.130 14.631 −9.832 1.00 10.94 C ATOM 713 C PRO A 96 −2.208 11.495 −8.667 1.00 11.30 C ATOM 714 O PRO A 96 −1.126 11.722 −9.242 1.00 11.03 O ATOM 715 N GLY A 97 −2.456 10.375 −7.996 1.00 11.13 N ATOM 716 CA GLY A 97 −1.457 9.323 −7.898 1.00 12.00 C ATOM 717 C GLY A 97 −1.037 8.875 −9.279 1.00 11.98 C ATOM 718 O GLY A 97 −1.829 8.921 −10.232 1.00 11.74 O ATOM 719 N THR A 98 0.220 8.452 −9.374 1.00 13.09 N ATOM 720 CA THR A 98 0.869 8.009 −10.626 1.00 13.70 C ATOM 721 CB THR A 98 0.148 6.814 −11.366 1.00 13.74 C ATOM 722 OG1 THR A 98 −1.010 7.283 −12.081 1.00 15.52 O ATOM 723 CG2 THR A 98 −0.243 5.728 −10.394 1.00 14.60 C ATOM 724 C THR A 98 1.139 9.149 −11.604 1.00 14.27 C ATOM 725 O THR A 98 1.608 8.908 −12.708 1.00 15.60 O ATOM 726 N THR A 99 0.832 10.389 −11.217 1.00 13.18 N ATOM 727 CA THR A 99 1.107 11.527 −12.102 1.00 12.89 C ATOM 728 CB THR A 99 −0.176 12.335 −12.512 1.00 13.67 C ATOM 729 OG1 THR A 99 −0.656 13.144 −11.429 1.00 12.87 O ATOM 730 CG2 THR A 99 −1.284 11.407 −13.040 1.00 14.02 C ATOM 731 C THR A 99 2.249 12.425 −11.582 1.00 12.99 C ATOM 732 O THR A 99 3.327 11.931 −11.280 1.00 13.12 O ATOM 733 N ALA A 100 2.033 13.734 −11.529 1.00 12.68 N ATOM 734 CA ALA A 100 3.090 14.657 −11.117 1.00 11.77 C ATOM 735 CB ALA A 100 4.032 14.965 −12.298 1.00 12.20 C ATOM 736 C ALA A 100 2.481 15.936 −10.575 1.00 11.63 C ATOM 737 O ALA A 100 1.296 16.215 −10.811 1.00 10.64 O ATOM 738 N ALA A 101 3.294 16.743 −9.889 1.00 11.30 N ATOM 739 CA ALA A 101 2.793 17.975 −9.291 1.00 11.18 C ATOM 740 CB ALA A 101 3.906 18.698 −8.518 1.00 10.54 C ATOM 741 C ALA A 101 2.143 18.922 −10.312 1.00 12.00 C ATOM 742 O ALA A 101 1.199 19.644 −9.981 1.00 12.35 O ATOM 743 N SER A 102 2.627 18.884 −11.557 1.00 13.14 N ATOM 744 CA SER A 102 2.049 19.712 −12.629 1.00 14.55 C ATOM 745 CB SER A 102 2.923 19.651 −13.887 1.00 15.27 C ATOM 746 OG SER A 102 3.029 18.337 −14.373 1.00 17.69 O ATOM 747 C SER A 102 0.581 19.383 −12.956 1.00 14.98 C ATOM 748 O SER A 102 −0.114 20.176 −13.603 1.00 14.30 O ATOM 749 N PHE A 103 0.106 18.229 −12.488 1.00 14.28 N ATOM 750 CA PHE A 103 −1.297 17.822 −12.675 1.00 13.69 C ATOM 751 CB PHE A 103 −1.379 16.327 −13.009 1.00 14.54 C ATOM 752 CG PHE A 103 −0.736 15.968 −14.313 1.00 15.30 C ATOM 753 CD1 PHE A 103 0.587 15.568 −14.366 1.00 17.34 C ATOM 754 CE1 PHE A 103 1.177 15.218 −15.581 1.00 19.81 C ATOM 755 CZ PHE A 103 0.435 15.284 −16.763 1.00 19.20 C ATOM 756 CE2 PHE A 103 −0.885 15.708 −16.724 1.00 21.07 C ATOM 757 CD2 PHE A 103 −1.469 16.031 −15.498 1.00 19.05 C ATOM 758 C PHE A 103 −2.197 18.144 −11.473 1.00 13.77 C ATOM 759 O PHE A 103 −3.398 17.874 −11.487 1.00 14.00 O ATOM 760 N GLY A 104 −1.603 18.716 −10.428 1.00 12.89 N ATOM 761 CA GLY A 104 −2.345 19.118 −9.239 1.00 13.71 C ATOM 762 C GLY A 104 −2.793 20.576 −9.280 1.00 13.46 C ATOM 763 O GLY A 104 −2.705 21.253 −10.309 1.00 14.84 O ATOM 764 N GLN A 105 −3.278 21.045 −8.140 1.00 13.60 N ATOM 765 CA GLN A 105 −3.740 22.417 −7.952 1.00 13.67 C ATOM 766 CB GLN A 105 −5.004 22.420 −7.067 1.00 15.08 C ATOM 767 CG GLN A 105 −5.539 23.801 −6.654 1.00 18.75 C ATOM 768 CD GLN A 105 −6.079 24.610 −7.822 1.00 24.42 C ATOM 769 OE1 GLN A 105 −6.881 24.113 −8.613 1.00 25.27 O ATOM 770 NE2 GLN A 105 −5.640 25.861 −7.935 1.00 24.80 N ATOM 771 C GLN A 105 −2.636 23.226 −7.285 1.00 13.40 C ATOM 772 O GLN A 105 −2.291 22.951 −6.140 1.00 13.14 O ATOM 773 N GLU A 106 −2.086 24.208 −7.994 1.00 13.06 N ATOM 774 CA GLU A 106 −1.093 25.088 −7.403 1.00 12.71 C ATOM 775 CB GLU A 106 −0.409 25.936 −8.474 1.00 12.64 C ATOM 776 CG GLU A 106 0.675 26.837 −7.918 1.00 14.18 C ATOM 777 CD GLU A 106 1.356 27.621 −9.005 1.00 14.34 C ATOM 778 OE1 GLU A 106 2.180 27.034 −9.731 1.00 15.78 O ATOM 779 OE2 GLU A 106 1.051 28.831 −9.145 1.00 17.06 O ATOM 780 C GLU A 106 −1.762 25.959 −6.356 1.00 12.69 C ATOM 781 O GLU A 106 −2.740 26.650 −6.642 1.00 13.76 O ATOM 782 N VAL A 107 −1.274 25.876 −5.127 1.00 11.35 N ATOM 783 CA VAL A 107 −1.785 26.718 −4.035 1.00 11.75 C ATOM 784 CB VAL A 107 −2.046 25.870 −2.783 1.00 11.64 C ATOM 785 CG1 VAL A 107 −2.493 26.751 −1.606 1.00 12.09 C ATOM 786 CG2 VAL A 107 −3.099 24.796 −3.090 1.00 13.27 C ATOM 787 C VAL A 107 −0.845 27.901 −3.772 1.00 11.57 C ATOM 788 O VAL A 107 −1.287 29.035 −3.574 1.00 12.74 O ATOM 789 N MET A 108 0.460 27.635 −3.771 1.00 10.59 N ATOM 790 CA MET A 108 1.477 28.679 −3.647 1.00 10.72 C ATOM 791 CB MET A 108 2.168 28.601 −2.283 1.00 10.85 C ATOM 792 CG MET A 108 1.242 28.882 −1.122 1.00 11.87 C ATOM 793 SD MET A 108 0.513 30.546 −1.129 1.00 14.91 S ATOM 794 CE MET A 108 1.796 31.455 −0.277 1.00 15.90 C ATOM 795 C MET A 108 2.492 28.419 −4.740 1.00 10.62 C ATOM 796 O MET A 108 3.038 27.321 −4.816 1.00 10.42 O ATOM 797 N SER A 109 2.737 29.400 −5.598 1.00 10.17 N ATOM 798 CA SER A 109 3.697 29.261 −6.696 1.00 10.51 C ATOM 799 CB SER A 109 3.684 30.517 −7.567 1.00 10.85 C ATOM 800 OG SER A 109 4.113 31.646 −6.818 1.00 13.34 O ATOM 801 C SER A 109 5.113 29.005 −6.182 1.00 10.03 C ATOM 802 O SER A 109 5.473 29.392 −5.072 1.00 9.10 O ATOM 803 N TYR A 110 5.930 28.358 −7.000 1.00 9.66 N ATOM 804 CA TYR A 110 7.311 28.074 −6.668 1.00 9.56 C ATOM 805 CB TYR A 110 7.888 27.131 −7.717 1.00 9.06 C ATOM 806 CG TYR A 110 9.304 26.656 −7.488 1.00 8.90 C ATOM 807 CD1 TYR A 110 10.387 27.334 −8.050 1.00 9.27 C ATOM 808 CE1 TYR A 110 11.692 26.870 −7.863 1.00 8.98 C ATOM 809 CZ TYR A 110 11.938 25.739 −7.106 1.00 8.80 C ATOM 810 OH TYR A 110 13.212 25.238 −6.923 1.00 9.40 O ATOM 811 CE2 TYR A 110 10.869 25.058 −6.532 1.00 9.65 C ATOM 812 CD2 TYR A 110 9.567 25.515 −6.738 1.00 8.74 C ATOM 813 C TYR A 110 8.144 29.345 −6.624 1.00 10.02 C ATOM 814 O TYR A 110 8.211 30.087 −7.629 1.00 11.20 O ATOM 815 N GLU A 111 8.790 29.562 −5.482 1.00 10.05 N ATOM 816 CA GLU A 111 9.757 30.652 −5.323 1.00 10.15 C ATOM 817 CB GLU A 111 9.556 31.362 −3.979 1.00 10.04 C ATOM 818 CG GLU A 111 10.546 32.507 −3.720 1.00 12.39 C ATOM 819 CD GLU A 111 10.300 33.234 −2.411 1.00 12.58 C ATOM 820 OE1 GLU A 111 9.129 33.313 −1.961 1.00 14.03 O ATOM 821 OE2 GLU A 111 11.302 33.717 −1.850 1.00 14.81 O ATOM 822 C GLU A 111 11.168 30.071 −5.380 1.00 10.67 C ATOM 823 O GLU A 111 11.472 29.109 −4.676 1.00 10.99 O ATOM 824 N SER A 112 12.051 30.664 −6.199 1.00 11.47 N ATOM 825 CA SER A 112 13.434 30.181 −6.328 1.00 12.27 C ATOM 826 CB SER A 112 14.216 31.078 −7.293 1.00 12.98 C ATOM 827 OG SER A 112 13.543 31.240 −8.518 1.00 14.60 O ATOM 828 C SER A 112 14.208 30.149 −5.011 1.00 12.28 C ATOM 829 O SER A 112 14.293 31.172 −4.326 1.00 13.14 O ATOM 830 N PRO A 113 14.807 29.005 −4.640 1.00 12.08 N ATOM 831 CA PRO A 113 15.747 29.046 −3.515 1.00 12.12 C ATOM 832 CB PRO A 113 16.194 27.587 −3.369 1.00 12.00 C ATOM 833 CG PRO A 113 15.056 26.791 −3.967 1.00 11.86 C ATOM 834 CD PRO A 113 14.597 27.623 −5.121 1.00 11.81 C ATOM 835 C PRO A 113 16.960 29.931 −3.798 1.00 13.02 C ATOM 836 O PRO A 113 17.624 29.776 −4.835 1.00 12.77 O ATOM 837 N ARG A 114 17.264 30.832 −2.864 1.00 12.67 N ATOM 838 CA ARG A 114 18.442 31.712 −2.969 1.00 14.34 C ATOM 839 CB ARG A 114 17.999 33.128 −3.318 1.00 15.11 C ATOM 840 CG ARG A 114 17.260 33.237 −4.656 1.00 18.32 C ATOM 841 CD ARG A 114 16.893 34.673 −4.969 1.00 26.56 C ATOM 842 NE ARG A 114 16.152 35.293 −3.878 1.00 31.69 N ATOM 843 CZ ARG A 114 14.829 35.258 −3.744 1.00 33.27 C ATOM 844 NH1 ARG A 114 14.072 34.625 −4.634 1.00 35.42 N ATOM 845 NH2 ARG A 114 14.262 35.855 −2.710 1.00 34.90 N ATOM 846 C ARG A 114 19.233 31.670 −1.651 1.00 13.74 C ATOM 847 O ARG A 114 19.269 32.650 −0.904 1.00 14.22 O ATOM 848 N PRO A 115 19.866 30.526 −1.344 1.00 13.88 N ATOM 849 CA PRO A 115 20.507 30.380 −0.033 1.00 13.86 C ATOM 850 CB PRO A 115 20.974 28.917 −0.035 1.00 14.45 C ATOM 851 CG PRO A 115 21.146 28.598 −1.489 1.00 14.04 C ATOM 852 CD PRO A 115 20.034 29.326 −2.175 1.00 13.73 C ATOM 853 C PRO A 115 21.718 31.300 0.126 1.00 14.67 C ATOM 854 O PRO A 115 22.486 31.487 −0.822 1.00 15.81 O ATOM 855 N THR A 116 21.846 31.890 1.306 1.00 14.57 N ATOM 856 CA THR A 116 23.075 32.568 1.727 1.00 15.62 C ATOM 857 CB THR A 116 22.992 34.107 1.613 1.00 15.85 C ATOM 858 OG1 THR A 116 22.159 34.617 2.659 1.00 16.51 O ATOM 859 CG2 THR A 116 22.463 34.566 0.247 1.00 14.60 C ATOM 860 C THR A 116 23.362 32.205 3.189 1.00 16.16 C ATOM 861 O THR A 116 22.523 31.608 3.901 1.00 15.19 O ATOM 862 N MET A 117 24.555 32.588 3.637 1.00 17.12 N ATOM 863 CA MET A 117 24.962 32.436 5.027 1.00 17.95 C ATOM 864 CB MET A 117 24.107 33.320 5.941 1.00 17.77 C ATOM 865 CG MET A 117 24.240 34.793 5.628 1.00 18.46 C ATOM 866 SD MET A 117 23.239 35.830 6.716 1.00 20.64 S ATOM 867 CE MET A 117 21.598 35.458 6.079 1.00 19.92 C ATOM 868 C MET A 117 24.915 30.997 5.495 1.00 17.54 C ATOM 869 O MET A 117 24.443 30.709 6.583 1.00 18.91 O ATOM 870 N GLY A 118 25.397 30.090 4.665 1.00 17.48 N ATOM 871 CA GLY A 118 25.508 28.706 5.068 1.00 16.34 C ATOM 872 C GLY A 118 24.693 27.821 4.159 1.00 15.94 C ATOM 873 O GLY A 118 24.062 28.296 3.218 1.00 16.77 O ATOM 874 N ILE A 119 24.740 26.532 4.449 1.00 16.04 N ATOM 875 CA ILE A 119 24.049 25.524 3.656 1.00 16.09 C ATOM 876 CB ILE A 119 24.773 24.160 3.784 1.00 16.48 C ATOM 877 CG1 ILE A 119 26.229 24.310 3.289 1.00 17.46 C ATOM 878 CD1 ILE A 119 27.123 23.119 3.562 1.00 17.99 C ATOM 879 CG2 ILE A 119 23.980 23.050 3.067 1.00 17.41 C ATOM 880 C ILE A 119 22.579 25.425 4.099 1.00 15.46 C ATOM 881 O ILE A 119 22.293 25.240 5.281 1.00 15.83 O ATOM 882 N HIS A 120 21.662 25.559 3.138 1.00 14.92 N ATOM 883 CA HIS A 120 20.217 25.451 3.391 1.00 13.59 C ATOM 884 CB HIS A 120 19.433 26.511 2.617 1.00 13.32 C ATOM 885 CG HIS A 120 19.646 27.916 3.083 1.00 13.00 C ATOM 886 ND1 HIS A 120 18.611 28.813 3.220 1.00 11.82 N ATOM 887 CE1 HIS A 120 19.089 29.973 3.630 1.00 13.67 C ATOM 888 NE2 HIS A 120 20.399 29.861 3.747 1.00 13.30 N ATOM 889 CD2 HIS A 120 20.774 28.588 3.415 1.00 13.44 C ATOM 890 C HIS A 120 19.701 24.103 2.905 1.00 13.48 C ATOM 891 O HIS A 120 20.095 23.625 1.836 1.00 13.83 O ATOM 892 N ARG A 121 18.813 23.505 3.679 1.00 12.73 N ATOM 893 CA ARG A 121 18.045 22.340 3.250 1.00 11.72 C ATOM 894 CB ARG A 121 17.688 21.472 4.449 1.00 11.82 C ATOM 895 CG ARG A 121 18.920 20.867 5.147 1.00 11.29 C ATOM 896 CD ARG A 121 18.594 20.404 6.524 1.00 13.52 C ATOM 897 NE ARG A 121 18.213 21.523 7.403 1.00 11.60 N ATOM 898 CZ ARG A 121 17.640 21.367 8.598 1.00 12.37 C ATOM 899 NH1 ARG A 121 17.367 20.156 9.075 1.00 15.43 N ATOM 900 NH2 ARG A 121 17.327 22.451 9.314 1.00 13.12 N ATOM 901 C ARG A 121 16.773 22.811 2.556 1.00 12.10 C ATOM 902 O ARG A 121 16.029 23.624 3.100 1.00 12.34 O ATOM 903 N LEU A 122 16.544 22.321 1.349 1.00 11.10 N ATOM 904 CA LEU A 122 15.266 22.541 0.657 1.00 10.59 C ATOM 905 CB LEU A 122 15.491 23.018 −0.773 1.00 11.01 C ATOM 906 CG LEU A 122 15.933 24.482 −0.935 1.00 9.29 C ATOM 907 CD1 LEU A 122 14.977 25.433 −0.176 1.00 11.83 C ATOM 908 CD2 LEU A 122 17.411 24.736 −0.525 1.00 12.76 C ATOM 909 C LEU A 122 14.514 21.226 0.692 1.00 10.81 C ATOM 910 O LEU A 122 14.963 20.224 0.124 1.00 10.75 O ATOM 911 N VAL A 123 13.400 21.223 1.408 1.00 9.83 N ATOM 912 CA VAL A 123 12.737 20.010 1.845 1.00 9.54 C ATOM 913 CB VAL A 123 12.494 20.036 3.395 1.00 9.97 C ATOM 914 CG1 VAL A 123 11.776 18.785 3.869 1.00 11.17 C ATOM 915 CG2 VAL A 123 13.815 20.228 4.182 1.00 10.12 C ATOM 916 C VAL A 123 11.400 19.859 1.131 1.00 9.63 C ATOM 917 O VAL A 123 10.573 20.765 1.180 1.00 9.57 O ATOM 918 N PHE A 124 11.197 18.705 0.492 1.00 9.73 N ATOM 919 CA PHE A 124 9.918 18.347 −0.145 1.00 9.69 C ATOM 920 CB PHE A 124 10.141 17.764 −1.536 1.00 9.63 C ATOM 921 CG PHE A 124 10.847 18.685 −2.462 1.00 7.46 C ATOM 922 CD1 PHE A 124 12.242 18.839 −2.416 1.00 8.40 C ATOM 923 CE1 PHE A 124 12.862 19.728 −3.292 1.00 10.48 C ATOM 924 CZ PHE A 124 12.102 20.466 −4.186 1.00 8.85 C ATOM 925 CE2 PHE A 124 10.741 20.321 −4.220 1.00 8.33 C ATOM 926 CD2 PHE A 124 10.116 19.455 −3.362 1.00 8.77 C ATOM 927 C PHE A 124 9.236 17.312 0.704 1.00 10.45 C ATOM 928 O PHE A 124 9.887 16.331 1.086 1.00 11.33 O ATOM 929 N VAL A 125 7.956 17.509 1.007 1.00 9.83 N ATOM 930 CA VAL A 125 7.191 16.596 1.863 1.00 10.12 C ATOM 931 CB VAL A 125 6.828 17.207 3.242 1.00 10.33 C ATOM 932 CG1 VAL A 125 6.264 16.123 4.181 1.00 10.69 C ATOM 933 CG2 VAL A 125 8.033 17.912 3.891 1.00 11.21 C ATOM 934 C VAL A 125 5.909 16.245 1.119 1.00 11.31 C ATOM 935 O VAL A 125 5.200 17.138 0.629 1.00 10.47 O ATOM 936 N LEU A 126 5.622 14.946 1.017 1.00 11.31 N ATOM 937 CA LEU A 126 4.429 14.477 0.317 1.00 13.02 C ATOM 938 CB LEU A 126 4.811 13.458 −0.760 1.00 13.12 C ATOM 939 CG LEU A 126 3.734 13.135 −1.797 1.00 13.99 C ATOM 940 CD1 LEU A 126 3.462 14.325 −2.725 1.00 11.96 C ATOM 941 CD2 LEU A 126 4.124 11.908 −2.618 1.00 13.59 C ATOM 942 C LEU A 126 3.475 13.863 1.315 1.00 12.82 C ATOM 943 O LEU A 126 3.893 13.033 2.128 1.00 12.99 O ATOM 944 N PHE A 127 2.220 14.289 1.274 1.00 12.69 N ATOM 945 CA PHE A 127 1.153 13.702 2.089 1.00 13.21 C ATOM 946 CB PHE A 127 0.478 14.770 2.943 1.00 13.79 C ATOM 947 CG PHE A 127 1.395 15.421 3.934 1.00 14.67 C ATOM 948 CD1 PHE A 127 1.573 14.863 5.197 1.00 16.53 C ATOM 949 CE1 PHE A 127 2.420 15.452 6.132 1.00 15.30 C ATOM 950 CZ PHE A 127 3.109 16.621 5.800 1.00 14.76 C ATOM 951 CE2 PHE A 127 2.933 17.190 4.533 1.00 15.46 C ATOM 952 CD2 PHE A 127 2.074 16.595 3.610 1.00 14.89 C ATOM 953 C PHE A 127 0.115 13.079 1.191 1.00 13.58 C ATOM 954 O PHE A 127 −0.165 13.571 0.098 1.00 12.54 O ATOM 955 N GLN A 128 −0.475 11.998 1.677 1.00 14.07 N ATOM 956 CA GLN A 128 −1.628 11.401 1.039 1.00 15.00 C ATOM 957 CB GLN A 128 −1.593 9.892 1.263 1.00 14.40 C ATOM 958 CG GLN A 128 −2.647 9.119 0.498 1.00 17.88 C ATOM 959 CD GLN A 128 −2.471 7.620 0.655 1.00 19.02 C ATOM 960 OE1 GLN A 128 −1.358 7.099 0.556 1.00 23.21 O ATOM 961 NE2 GLN A 128 −3.568 6.917 0.896 1.00 22.83 N ATOM 962 C GLN A 128 −2.898 11.984 1.647 1.00 14.63 C ATOM 963 O GLN A 128 −3.084 11.961 2.868 1.00 16.63 O ATOM 964 N GLN A 129 −3.763 12.525 0.797 1.00 15.09 N ATOM 965 CA GLN A 129 −5.054 13.067 1.206 1.00 14.67 C ATOM 966 CB GLN A 129 −5.546 14.050 0.154 1.00 15.37 C ATOM 967 CG GLN A 129 −4.582 15.224 −0.057 1.00 13.79 C ATOM 968 CD GLN A 129 −4.934 16.116 −1.247 1.00 13.40 C ATOM 969 OE1 GLN A 129 −4.049 16.816 −1.769 1.00 13.01 O ATOM 970 NE2 GLN A 129 −6.204 16.101 −1.698 1.00 11.64 N ATOM 971 C GLN A 129 −6.083 11.944 1.370 1.00 16.55 C ATOM 972 O GLN A 129 −5.941 10.873 0.766 1.00 16.37 O ATOM 973 N LEU A 130 −7.109 12.189 2.183 1.00 18.50 N ATOM 974 CA LEU A 130 −8.207 11.207 2.287 1.00 19.97 C ATOM 975 CB LEU A 130 −9.097 11.463 3.517 1.00 19.90 C ATOM 976 CG LEU A 130 −8.622 10.883 4.858 1.00 22.65 C ATOM 977 CD1 LEU A 130 −9.673 11.088 5.947 1.00 23.26 C ATOM 978 CD2 LEU A 130 −8.245 9.400 4.777 1.00 24.56 C ATOM 979 C LEU A 130 −9.055 11.182 1.022 1.00 20.25 C ATOM 980 O LEU A 130 −9.735 10.196 0.749 1.00 20.92 O ATOM 981 N GLY A 131 −9.024 12.284 0.277 1.00 20.14 N ATOM 982 CA GLY A 131 −9.701 12.458 −1.015 1.00 19.37 C ATOM 983 C GLY A 131 −9.372 13.880 −1.483 1.00 19.58 C ATOM 984 O GLY A 131 −8.575 14.563 −0.841 1.00 18.85 O ATOM 985 N ARG A 132 −9.980 14.334 −2.580 1.00 19.45 N ATOM 986 CA ARG A 132 −9.835 15.728 −3.012 1.00 20.28 C ATOM 987 CB ARG A 132 −10.724 16.049 −4.214 1.00 20.91 C ATOM 988 CG ARG A 132 −10.174 15.577 −5.543 1.00 23.26 C ATOM 989 CD ARG A 132 −10.734 16.402 −6.688 1.00 27.64 C ATOM 990 NE ARG A 132 −10.051 16.107 −7.946 1.00 30.86 N ATOM 991 CZ ARG A 132 −10.573 15.390 −8.940 1.00 32.62 C ATOM 992 NH1 ARG A 132 −11.804 14.898 −8.847 1.00 33.66 N ATOM 993 NH2 ARG A 132 −9.868 15.180 −10.042 1.00 32.84 N ATOM 994 C ARG A 132 −10.222 16.625 −1.857 1.00 19.94 C ATOM 995 O ARG A 132 −11.180 16.343 −1.130 1.00 20.21 O ATOM 996 N GLN A 133 −9.456 17.686 −1.657 1.00 19.12 N ATOM 997 CA GLN A 133 −9.789 18.599 −0.589 1.00 19.38 C ATOM 998 CB GLN A 133 −9.241 18.113 0.757 1.00 20.24 C ATOM 999 CG GLN A 133 −7.767 18.277 0.967 1.00 21.09 C ATOM 1000 CD GLN A 133 −7.299 17.544 2.211 1.00 23.17 C ATOM 1001 OE1 GLN A 133 −6.610 16.531 2.113 1.00 24.79 O ATOM 1002 NE2 GLN A 133 −7.697 18.034 3.389 1.00 23.32 N ATOM 1003 C GLN A 133 −9.389 20.016 −0.903 1.00 18.56 C ATOM 1004 O GLN A 133 −8.573 20.275 −1.789 1.00 19.53 O ATOM 1005 N THR A 134 −10.002 20.930 −0.161 1.00 18.00 N ATOM 1006 CA THR A 134 −9.748 22.348 −0.315 1.00 17.42 C ATOM 1007 CB THR A 134 −11.033 23.181 −0.105 1.00 17.19 C ATOM 1008 OG1 THR A 134 −12.032 22.768 −1.048 1.00 17.69 O ATOM 1009 CG2 THR A 134 −10.748 24.690 −0.273 1.00 16.85 C ATOM 1010 C THR A 134 −8.693 22.765 0.693 1.00 16.68 C ATOM 1011 O THR A 134 −8.889 22.695 1.903 1.00 17.87 O ATOM 1012 N VAL A 135 −7.573 23.213 0.162 1.00 15.65 N ATOM 1013 CA VAL A 135 −6.499 23.747 0.955 1.00 15.37 C ATOM 1014 CB VAL A 135 −5.186 22.988 0.688 1.00 15.83 C ATOM 1015 CG1 VAL A 135 −4.030 23.678 1.386 1.00 15.56 C ATOM 1016 CG2 VAL A 135 −5.320 21.544 1.140 1.00 16.93 C ATOM 1017 C VAL A 135 −6.368 25.226 0.587 1.00 15.08 C ATOM 1018 O VAL A 135 −6.070 25.572 −0.560 1.00 16.30 O ATOM 1019 N TYR A 136 −6.605 26.094 1.571 1.00 14.42 N ATOM 1020 CA TYR A 136 −6.497 27.522 1.364 1.00 14.68 C ATOM 1021 CB TYR A 136 −7.226 28.290 2.477 1.00 14.50 C ATOM 1022 CG TYR A 136 −8.715 28.024 2.534 1.00 14.01 C ATOM 1023 CD1 TYR A 136 −9.245 26.984 3.303 1.00 12.67 C ATOM 1024 CE1 TYR A 136 −10.627 26.755 3.351 1.00 13.07 C ATOM 1025 CZ TYR A 136 −11.480 27.582 2.622 1.00 12.92 C ATOM 1026 OH TYR A 136 −12.844 27.371 2.655 1.00 14.02 O ATOM 1027 CE2 TYR A 136 −10.964 28.606 1.844 1.00 14.17 C ATOM 1028 CD2 TYR A 136 −9.596 28.823 1.808 1.00 14.69 C ATOM 1029 C TYR A 136 −5.039 27.935 1.327 1.00 14.65 C ATOM 1030 O TYR A 136 −4.216 27.443 2.106 1.00 15.76 O ATOM 1031 N ALA A 137 −4.724 28.845 0.418 1.00 13.18 N ATOM 1032 CA ALA A 137 −3.364 29.399 0.357 1.00 12.67 C ATOM 1033 CB ALA A 137 −3.242 30.381 −0.785 1.00 12.89 C ATOM 1034 C ALA A 137 −3.011 30.093 1.678 1.00 12.48 C ATOM 1035 O ALA A 137 −3.762 30.948 2.148 1.00 12.38 O ATOM 1036 N PRO A 138 −1.873 29.734 2.294 1.00 12.25 N ATOM 1037 CA PRO A 138 −1.389 30.479 3.445 1.00 12.45 C ATOM 1038 CB PRO A 138 0.023 29.905 3.642 1.00 12.65 C ATOM 1039 CG PRO A 138 −0.158 28.475 3.235 1.00 12.72 C ATOM 1040 CD PRO A 138 −0.994 28.582 1.993 1.00 12.44 C ATOM 1041 C PRO A 138 −1.331 31.964 3.132 1.00 12.13 C ATOM 1042 O PRO A 138 −1.088 32.335 1.981 1.00 13.03 O ATOM 1043 N GLY A 139 −1.599 32.796 4.136 1.00 12.36 N ATOM 1044 CA GLY A 139 −1.580 34.241 3.947 1.00 12.46 C ATOM 1045 C GLY A 139 −0.193 34.840 3.792 1.00 12.60 C ATOM 1046 O GLY A 139 −0.050 35.987 3.403 1.00 13.35 O ATOM 1047 N TRP A 140 0.839 34.053 4.104 1.00 12.05 N ATOM 1048 CA TRP A 140 2.243 34.482 4.004 1.00 11.38 C ATOM 1049 CB TRP A 140 2.613 35.436 5.159 1.00 11.60 C ATOM 1050 CG TRP A 140 2.293 34.856 6.509 1.00 12.07 C ATOM 1051 CD1 TRP A 140 3.077 34.002 7.246 1.00 11.39 C ATOM 1052 NE1 TRP A 140 2.441 33.650 8.402 1.00 12.72 N ATOM 1053 CE2 TRP A 140 1.213 34.256 8.446 1.00 11.61 C ATOM 1054 CD2 TRP A 140 1.077 35.015 7.255 1.00 10.56 C ATOM 1055 CE3 TRP A 140 −0.099 35.753 7.054 1.00 12.34 C ATOM 1056 CZ3 TRP A 140 −1.090 35.704 8.021 1.00 12.25 C ATOM 1057 CH2 TRP A 140 −0.932 34.929 9.196 1.00 12.88 C ATOM 1058 CZ2 TRP A 140 0.214 34.207 9.429 1.00 12.38 C ATOM 1059 C TRP A 140 3.102 33.222 4.023 1.00 10.15 C ATOM 1060 O TRP A 140 2.653 32.160 4.460 1.00 10.45 O ATOM 1061 N ARG A 141 4.327 33.347 3.542 1.00 9.66 N ATOM 1062 CA ARG A 141 5.212 32.198 3.400 1.00 8.72 C ATOM 1063 CB ARG A 141 6.017 32.335 2.110 1.00 8.68 C ATOM 1064 CG ARG A 141 5.188 32.246 0.838 1.00 9.36 C ATOM 1065 CD ARG A 141 6.057 32.535 −0.355 1.00 11.08 C ATOM 1066 NE ARG A 141 5.326 32.504 −1.622 1.00 10.70 N ATOM 1067 CZ ARG A 141 5.378 31.523 −2.526 1.00 9.22 C ATOM 1068 NH1 ARG A 141 6.131 30.443 −2.325 1.00 9.69 N ATOM 1069 NH2 ARG A 141 4.687 31.634 −3.650 1.00 10.33 N ATOM 1070 C ARG A 141 6.144 31.956 4.586 1.00 8.52 C ATOM 1071 O ARG A 141 6.623 30.830 4.794 1.00 8.51 O ATOM 1072 N GLN A 142 6.446 32.987 5.352 1.00 8.87 N ATOM 1073 CA GLN A 142 7.421 32.823 6.422 1.00 9.56 C ATOM 1074 CB GLN A 142 8.093 34.152 6.782 1.00 11.20 C ATOM 1075 CG GLN A 142 7.277 35.053 7.669 1.00 13.08 C ATOM 1076 CD GLN A 142 6.243 35.866 6.947 1.00 12.46 C ATOM 1077 OE1 GLN A 142 6.078 35.794 5.716 1.00 13.85 O ATOM 1078 NE2 GLN A 142 5.531 36.680 7.724 1.00 16.54 N ATOM 1079 C GLN A 142 6.800 32.159 7.650 1.00 9.75 C ATOM 1080 O GLN A 142 5.578 32.242 7.875 1.00 9.56 O ATOM 1081 N ASN A 143 7.634 31.489 8.439 1.00 9.63 N ATOM 1082 CA ASN A 143 7.174 30.875 9.689 1.00 9.86 C ATOM 1083 CB ASN A 143 6.876 31.964 10.749 1.00 10.64 C ATOM 1084 CG ASN A 143 8.100 32.796 11.086 1.00 11.63 C ATOM 1085 OD1 ASN A 143 8.095 34.002 10.910 1.00 15.35 O ATOM 1086 ND2 ASN A 143 9.169 32.143 11.537 1.00 12.42 N ATOM 1087 C ASN A 143 5.983 29.936 9.476 1.00 10.55 C ATOM 1088 O ASN A 143 5.035 29.881 10.276 1.00 11.27 O ATOM 1089 N PHE A 144 6.034 29.231 8.348 1.00 9.68 N ATOM 1090 CA PHE A 144 5.137 28.103 8.076 1.00 8.73 C ATOM 1091 CB PHE A 144 5.272 27.713 6.595 1.00 9.14 C ATOM 1092 CG PHE A 144 4.309 26.648 6.114 1.00 8.97 C ATOM 1093 CD1 PHE A 144 3.004 26.973 5.770 1.00 8.19 C ATOM 1094 CE1 PHE A 144 2.126 25.974 5.296 1.00 9.14 C ATOM 1095 CZ PHE A 144 2.587 24.678 5.139 1.00 9.36 C ATOM 1096 CE2 PHE A 144 3.900 24.359 5.472 1.00 8.56 C ATOM 1097 CD2 PHE A 144 4.737 25.330 5.964 1.00 9.09 C ATOM 1098 C PHE A 144 5.506 26.932 8.994 1.00 9.05 C ATOM 1099 O PHE A 144 6.645 26.823 9.474 1.00 9.38 O ATOM 1100 N ASN A 145 4.535 26.059 9.247 1.00 9.65 N ATOM 1101 CA ASN A 145 4.789 24.883 10.064 1.00 10.23 C ATOM 1102 CB ASN A 145 4.346 25.096 11.515 1.00 10.90 C ATOM 1103 CG ASN A 145 4.847 24.012 12.409 1.00 12.57 C ATOM 1104 OD1 ASN A 145 4.290 22.915 12.440 1.00 12.21 O ATOM 1105 ND2 ASN A 145 5.936 24.284 13.115 1.00 14.68 N ATOM 1106 C ASN A 145 4.102 23.685 9.459 1.00 10.44 C ATOM 1107 O ASN A 145 2.880 23.566 9.516 1.00 10.47 O ATOM 1108 N THR A 146 4.905 22.797 8.881 1.00 9.97 N ATOM 1109 CA THR A 146 4.387 21.642 8.148 1.00 10.67 C ATOM 1110 CB THR A 146 5.530 20.871 7.481 1.00 10.45 C ATOM 1111 OG1 THR A 146 6.124 21.716 6.493 1.00 10.30 O ATOM 1112 CG2 THR A 146 5.028 19.626 6.761 1.00 10.92 C ATOM 1113 C THR A 146 3.551 20.737 9.043 1.00 11.55 C ATOM 1114 O THR A 146 2.496 20.249 8.611 1.00 12.15 O ATOM 1115 N LYS A 147 3.993 20.560 10.288 1.00 12.41 N ATOM 1116 CA LYS A 147 3.218 19.752 11.254 1.00 13.55 C ATOM 1117 CB LYS A 147 3.987 19.575 12.550 1.00 14.23 C ATOM 1118 CG LYS A 147 5.138 18.616 12.473 1.00 17.93 C ATOM 1119 CD LYS A 147 5.862 18.590 13.810 1.00 21.51 C ATOM 1120 CE LYS A 147 7.079 17.707 13.760 1.00 23.93 C ATOM 1121 NZ LYS A 147 7.807 17.739 15.066 1.00 26.40 N ATOM 1122 C LYS A 147 1.860 20.386 11.534 1.00 13.72 C ATOM 1123 O LYS A 147 0.832 19.705 11.512 1.00 14.53 O ATOM 1124 N ASP A 148 1.823 21.687 11.786 1.00 13.45 N ATOM 1125 CA ASP A 148 0.547 22.369 12.030 1.00 13.94 C ATOM 1126 CB ASP A 148 0.763 23.832 12.406 1.00 14.55 C ATOM 1127 CG ASP A 148 1.469 24.000 13.748 1.00 15.94 C ATOM 1128 OD1 ASP A 148 1.539 23.014 14.527 1.00 18.07 O ATOM 1129 OD2 ASP A 148 1.964 25.129 13.982 1.00 19.43 O ATOM 1130 C ASP A 148 −0.356 22.298 10.800 1.00 14.52 C ATOM 1131 O ASP A 148 −1.569 22.124 10.920 1.00 15.55 O ATOM 1132 N PHE A 149 0.239 22.455 9.615 1.00 13.72 N ATOM 1133 CA PHE A 149 −0.484 22.427 8.350 1.00 13.57 C ATOM 1134 CB PHE A 149 0.499 22.751 7.217 1.00 12.47 C ATOM 1135 CG PHE A 149 −0.055 22.561 5.832 1.00 11.03 C ATOM 1136 CD1 PHE A 149 −0.871 23.519 5.278 1.00 11.99 C ATOM 1137 CE1 PHE A 149 −1.374 23.361 3.986 1.00 11.02 C ATOM 1138 CZ PHE A 149 −1.058 22.221 3.258 1.00 11.51 C ATOM 1139 CE2 PHE A 149 −0.244 21.266 3.802 1.00 12.90 C ATOM 1140 CD2 PHE A 149 0.261 21.431 5.086 1.00 12.02 C ATOM 1141 C PHE A 149 −1.149 21.064 8.138 1.00 14.01 C ATOM 1142 O PHE A 149 −2.322 20.998 7.779 1.00 13.74 O ATOM 1143 N ALA A 150 −0.397 19.990 8.359 1.00 15.01 N ATOM 1144 CA ALA A 150 −0.919 18.629 8.173 1.00 15.77 C ATOM 1145 CB ALA A 150 0.173 17.609 8.285 1.00 15.18 C ATOM 1146 C ALA A 150 −2.040 18.343 9.165 1.00 16.79 C ATOM 1147 O ALA A 150 −3.004 17.642 8.835 1.00 17.16 O ATOM 1148 N GLU A 151 −1.919 18.897 10.367 1.00 17.48 N ATOM 1149 CA GLU A 151 −2.934 18.778 11.400 1.00 18.12 C ATOM 1150 CB GLU A 151 −2.417 19.372 12.714 1.00 18.66 C ATOM 1151 CG GLU A 151 −3.394 19.300 13.882 1.00 19.71 C ATOM 1152 CD GLU A 151 −3.723 17.876 14.291 1.00 22.61 C ATOM 1153 OE1 GLU A 151 −2.793 17.056 14.425 1.00 25.66 O ATOM 1154 OE2 GLU A 151 −4.923 17.588 14.479 1.00 23.68 O ATOM 1155 C GLU A 151 −4.216 19.458 10.951 1.00 18.07 C ATOM 1156 O GLU A 151 −5.280 18.829 10.983 1.00 18.28 O ATOM 1157 N LEU A 152 −4.112 20.716 10.506 1.00 17.59 N ATOM 1158 CA LEU A 152 −5.238 21.508 10.008 1.00 17.94 C ATOM 1159 CB LEU A 152 −4.766 22.887 9.508 1.00 17.76 C ATOM 1160 CG LEU A 152 −5.807 23.977 9.192 1.00 18.37 C ATOM 1161 CD1 LEU A 152 −6.685 24.308 10.420 1.00 21.33 C ATOM 1162 CD2 LEU A 152 −5.146 25.237 8.667 1.00 18.72 C ATOM 1163 C LEU A 152 −6.014 20.761 8.922 1.00 17.06 C ATOM 1164 O LEU A 152 −7.251 20.691 8.972 1.00 18.52 O ATOM 1165 N TYR A 153 −5.294 20.149 7.985 1.00 16.96 N ATOM 1166 CA TYR A 153 −5.924 19.549 6.809 1.00 16.72 C ATOM 1167 CB TYR A 153 −5.210 20.034 5.548 1.00 16.28 C ATOM 1168 CG TYR A 153 −5.463 21.496 5.309 1.00 14.51 C ATOM 1169 CD1 TYR A 153 −6.722 21.942 4.906 1.00 13.96 C ATOM 1170 CE1 TYR A 153 −6.988 23.292 4.692 1.00 14.15 C ATOM 1171 CZ TYR A 153 −5.987 24.238 4.887 1.00 14.55 C ATOM 1172 OH TYR A 153 −6.263 25.579 4.657 1.00 15.55 O ATOM 1173 CE2 TYR A 153 −4.722 23.823 5.299 1.00 14.25 C ATOM 1174 CD2 TYR A 153 −4.463 22.451 5.509 1.00 13.74 C ATOM 1175 C TYR A 153 −6.054 18.019 6.836 1.00 17.52 C ATOM 1176 O TYR A 153 −6.398 17.402 5.823 1.00 17.05 O ATOM 1177 N ASN A 154 −5.793 17.420 8.001 1.00 18.63 N ATOM 1178 CA ASN A 154 −5.824 15.953 8.167 1.00 18.72 C ATOM 1179 CB ASN A 154 −7.270 15.436 8.278 1.00 19.28 C ATOM 1180 CG ASN A 154 −7.341 13.994 8.742 1.00 21.31 C ATOM 1181 OD1 ASN A 154 −6.435 13.496 9.412 1.00 21.09 O ATOM 1182 ND2 ASN A 154 −8.416 13.307 8.367 1.00 23.10 N ATOM 1183 C ASN A 154 −5.050 15.201 7.081 1.00 19.12 C ATOM 1184 O ASN A 154 −5.574 14.307 6.404 1.00 19.41 O ATOM 1185 N LEU A 155 −3.790 15.570 6.926 1.00 18.73 N ATOM 1186 CA LEU A 155 −2.933 14.949 5.939 1.00 19.64 C ATOM 1187 CB LEU A 155 −1.990 16.001 5.355 1.00 19.37 C ATOM 1188 CG LEU A 155 −2.720 17.114 4.598 1.00 19.63 C ATOM 1189 CD1 LEU A 155 −1.735 18.155 4.151 1.00 17.67 C ATOM 1190 CD2 LEU A 155 −3.470 16.550 3.398 1.00 19.42 C ATOM 1191 C LEU A 155 −2.155 13.777 6.517 1.00 19.88 C ATOM 1192 O LEU A 155 −1.414 13.111 5.791 1.00 20.91 O ATOM 1193 N GLY A 156 −2.325 13.541 7.820 1.00 20.53 N ATOM 1194 CA GLY A 156 −1.637 12.458 8.532 1.00 20.50 C ATOM 1195 C GLY A 156 −0.141 12.671 8.603 1.00 20.23 C ATOM 1196 O GLY A 156 0.318 13.812 8.697 1.00 21.15 O ATOM 1197 N SER A 157 0.623 11.581 8.560 1.00 19.83 N ATOM 1198 CA SER A 157 2.083 11.649 8.507 1.00 19.53 C ATOM 1199 CB SER A 157 2.710 10.510 9.325 1.00 19.37 C ATOM 1200 OG SER A 157 2.170 9.252 8.950 1.00 21.33 O ATOM 1201 C SER A 157 2.564 11.582 7.060 1.00 18.45 C ATOM 1202 O SER A 157 1.866 11.030 6.196 1.00 18.67 O ATOM 1203 N PRO A 158 3.752 12.143 6.770 1.00 17.29 N ATOM 1204 CA PRO A 158 4.268 12.102 5.398 1.00 16.75 C ATOM 1205 CB PRO A 158 5.699 12.610 5.546 1.00 16.44 C ATOM 1206 CG PRO A 158 5.665 13.478 6.758 1.00 16.01 C ATOM 1207 CD PRO A 158 4.647 12.885 7.680 1.00 17.00 C ATOM 1208 C PRO A 158 4.289 10.686 4.811 1.00 16.87 C ATOM 1209 O PRO A 158 4.533 9.703 5.537 1.00 17.66 O ATOM 1210 N VAL A 159 4.007 10.574 3.521 1.00 16.32 N ATOM 1211 CA VAL A 159 4.219 9.317 2.812 1.00 15.92 C ATOM 1212 CB VAL A 159 3.097 9.019 1.808 1.00 16.62 C ATOM 1213 CG1 VAL A 159 1.790 8.823 2.538 1.00 16.77 C ATOM 1214 CG2 VAL A 159 2.996 10.117 0.717 1.00 14.85 C ATOM 1215 C VAL A 159 5.589 9.266 2.144 1.00 15.38 C ATOM 1216 O VAL A 159 6.068 8.197 1.762 1.00 15.63 O ATOM 1217 N ALA A 160 6.229 10.435 2.016 1.00 13.87 N ATOM 1218 CA ALA A 160 7.543 10.547 1.428 1.00 13.29 C ATOM 1219 CB ALA A 160 7.479 10.356 −0.079 1.00 14.31 C ATOM 1220 C ALA A 160 8.129 11.913 1.746 1.00 13.11 C ATOM 1221 O ALA A 160 7.396 12.873 1.971 1.00 13.88 O ATOM 1222 N ALA A 161 9.447 11.982 1.737 1.00 13.56 N ATOM 1223 CA ALA A 161 10.127 13.268 1.794 1.00 14.00 C ATOM 1224 CB ALA A 161 10.120 13.854 3.224 1.00 13.91 C ATOM 1225 C ALA A 161 11.538 13.152 1.266 1.00 14.79 C ATOM 1226 O ALA A 161 12.145 12.078 1.330 1.00 15.53 O ATOM 1227 N VAL A 162 12.072 14.247 0.749 1.00 14.96 N ATOM 1228 CA VAL A 162 13.433 14.273 0.254 1.00 14.10 C ATOM 1229 CB VAL A 162 13.533 13.713 −1.190 1.00 14.46 C ATOM 1230 CG1 VAL A 162 13.041 14.726 −2.200 1.00 13.96 C ATOM 1231 CG2 VAL A 162 14.947 13.206 −1.535 1.00 14.31 C ATOM 1232 C VAL A 162 13.916 15.699 0.392 1.00 14.11 C ATOM 1233 O VAL A 162 13.114 16.638 0.417 1.00 12.90 O ATOM 1234 N TYR A 163 15.211 15.886 0.541 1.00 14.12 N ATOM 1235 CA TYR A 163 15.731 17.231 0.567 1.00 14.32 C ATOM 1236 CB TYR A 163 15.934 17.727 2.009 1.00 14.45 C ATOM 1237 CG TYR A 163 17.152 17.144 2.711 1.00 14.76 C ATOM 1238 CD1 TYR A 163 18.326 17.869 2.817 1.00 15.77 C ATOM 1239 CE1 TYR A 163 19.460 17.312 3.456 1.00 14.86 C ATOM 1240 CZ TYR A 163 19.379 16.047 3.987 1.00 15.49 C ATOM 1241 OH TYR A 163 20.477 15.477 4.620 1.00 17.60 O ATOM 1242 CE2 TYR A 163 18.218 15.319 3.892 1.00 15.78 C ATOM 1243 CD2 TYR A 163 17.113 15.865 3.259 1.00 15.86 C ATOM 1244 C TYR A 163 17.028 17.310 −0.192 1.00 14.67 C ATOM 1245 O TYR A 163 17.756 16.317 −0.287 1.00 15.31 O ATOM 1246 N PHE A 164 17.319 18.481 −0.720 1.00 14.09 N ATOM 1247 CA PHE A 164 18.651 18.772 −1.196 1.00 13.71 C ATOM 1248 CB PHE A 164 18.675 19.063 −2.703 1.00 13.88 C ATOM 1249 CG PHE A 164 17.966 20.310 −3.124 1.00 13.15 C ATOM 1250 CD1 PHE A 164 16.637 20.267 −3.521 1.00 12.93 C ATOM 1251 CE1 PHE A 164 15.995 21.427 −3.968 1.00 10.81 C ATOM 1252 CZ PHE A 164 16.686 22.628 −4.039 1.00 12.04 C ATOM 1253 CE2 PHE A 164 18.029 22.674 −3.634 1.00 12.05 C ATOM 1254 CD2 PHE A 164 18.653 21.518 −3.211 1.00 12.96 C ATOM 1255 C PHE A 164 19.289 19.863 −0.358 1.00 14.36 C ATOM 1256 O PHE A 164 18.598 20.613 0.346 1.00 13.83 O ATOM 1257 N ASN A 165 20.612 19.920 −0.395 1.00 15.33 N ATOM 1258 CA ASN A 165 21.339 21.024 0.193 1.00 16.05 C ATOM 1259 CB ASN A 165 22.604 20.506 0.879 1.00 16.27 C ATOM 1260 CG ASN A 165 22.324 19.734 2.154 1.00 16.91 C ATOM 1261 OD1 ASN A 165 22.900 18.653 2.381 1.00 20.15 O ATOM 1262 ND2 ASN A 165 21.451 20.267 3.006 1.00 16.17 N ATOM 1263 C ASN A 165 21.719 22.024 −0.887 1.00 16.67 C ATOM 1264 O ASN A 165 22.028 21.642 −2.027 1.00 16.92 O ATOM 1265 N SER A 166 21.719 23.306 −0.541 1.00 16.19 N ATOM 1266 CA SER A 166 22.118 24.361 −1.453 1.00 16.96 C ATOM 1267 CB SER A 166 20.909 24.890 −2.255 1.00 16.85 C ATOM 1268 OG SER A 166 21.302 25.638 −3.398 1.00 16.80 O ATOM 1269 C SER A 166 22.779 25.489 −0.665 1.00 17.69 C ATOM 1270 O SER A 166 22.361 25.816 0.453 1.00 16.27 O ATOM 1271 N GLN A 167 23.815 26.070 −1.250 1.00 18.62 N ATOM 1272 CA GLN A 167 24.455 27.255 −0.688 1.00 20.82 C ATOM 1273 CB GLN A 167 25.731 26.875 0.077 1.00 20.52 C ATOM 1274 CG GLN A 167 26.788 26.159 −0.749 1.00 21.67 C ATOM 1275 CD GLN A 167 27.973 25.753 0.083 1.00 22.50 C ATOM 1276 OE1 GLN A 167 28.563 26.576 0.776 1.00 26.39 O ATOM 1277 NE2 GLN A 167 28.326 24.471 0.035 1.00 24.55 N ATOM 1278 C GLN A 167 24.738 28.248 −1.810 1.00 21.81 C ATOM 1279 O GLN A 167 24.673 27.888 −2.994 1.00 21.58 O ATOM 1280 N ARG A 168 25.034 29.494 −1.443 1.00 23.44 N ATOM 1281 CA ARG A 168 25.360 30.530 −2.412 1.00 25.87 C ATOM 1282 CB ARG A 168 25.821 31.796 −1.686 1.00 25.72 C ATOM 1283 CG ARG A 168 25.798 33.042 −2.555 1.00 28.37 C ATOM 1284 CD ARG A 168 26.821 34.067 −2.092 1.00 32.14 C ATOM 1285 NE ARG A 168 26.464 34.700 −0.828 1.00 34.57 N ATOM 1286 CZ ARG A 168 25.763 35.827 −0.722 1.00 35.59 C ATOM 1287 NH1 ARG A 168 25.318 36.448 −1.808 1.00 36.78 N ATOM 1288 NH2 ARG A 168 25.492 36.322 0.476 1.00 36.00 N ATOM 1289 C ARG A 168 26.462 30.062 −3.369 1.00 27.24 C ATOM 1290 O ARG A 168 27.385 29.359 −2.959 1.00 27.83 O ATOM 1291 N GLU A 169 26.349 30.452 −4.633 1.00 29.12 N ATOM 1292 CA GLU A 169 27.375 30.149 −5.627 1.00 31.26 C ATOM 1293 CB GLU A 169 26.796 30.192 −7.042 1.00 31.49 C ATOM 1294 CG GLU A 169 26.098 28.916 −7.452 1.00 33.07 C ATOM 1295 CD GLU A 169 25.369 29.052 −8.774 1.00 34.47 C ATOM 1296 OE1 GLU A 169 26.041 29.085 −9.832 1.00 36.26 O ATOM 1297 OE2 GLU A 169 24.121 29.115 −8.755 1.00 31.69 O ATOM 1298 C GLU A 169 28.532 31.125 −5.501 1.00 31.98 C ATOM 1299 O GLU A 169 29.645 30.730 −5.147 1.00 33.31 O ATOM 1300 N ALA A 170 28.250 32.398 −5.766 1.00 33.08 N ATOM 1301 CA ALA A 170 29.251 33.461 −5.678 1.00 34.02 C ATOM 1302 CB ALA A 170 28.911 34.603 −6.654 1.00 34.26 C ATOM 1303 C ALA A 170 29.391 33.974 −4.242 1.00 34.33 C ATOM 1304 O ALA A 170 29.372 35.182 −3.981 1.00 35.14 O ATOM 1305 O HOH C 1 −5.319 32.872 0.892 1.00 11.89 O ATOM 1306 O HOH C 2 19.656 34.008 3.192 1.00 16.18 O ATOM 1307 O HOH C 3 −0.775 34.312 0.142 1.00 18.33 O ATOM 1308 O HOH C 4 13.932 32.961 −2.215 1.00 18.05 O ATOM 1309 O HOH C 5 5.804 35.827 10.784 1.00 23.16 O ATOM 1310 O HOH C 6 3.485 30.700 6.667 1.00 10.31 O ATOM 1311 O HOH C 7 6.458 20.754 −11.543 1.00 13.49 O ATOM 1312 O HOH C 8 4.462 1.175 0.495 1.00 39.86 O ATOM 1313 O HOH C 9 6.820 21.202 10.936 1.00 14.11 O ATOM 1314 O HOH C 10 1.813 27.035 9.251 1.00 15.37 O ATOM 1315 O HOH C 11 5.640 18.117 −11.676 1.00 12.45 O ATOM 1316 O HOH C 12 9.882 11.451 15.590 1.00 16.54 O ATOM 1317 O HOH C 13 12.428 12.848 16.110 1.00 20.36 O ATOM 1318 O HOH C 14 −10.592 22.653 3.869 1.00 22.72 O ATOM 1319 O HOH C 15 6.929 21.179 −14.056 1.00 20.59 O ATOM 1320 O HOH C 16 −12.267 20.234 −2.432 1.00 29.90 O ATOM 1321 O HOH C 17 1.042 31.761 −5.540 1.00 19.32 O ATOM 1322 O HOH C 18 −6.982 10.623 −1.828 1.00 16.84 O ATOM 1323 O HOH C 19 26.045 16.162 −3.164 1.00 27.74 O ATOM 1324 O HOH C 20 9.550 35.948 11.972 1.00 21.37 O ATOM 1325 O HOH C 21 22.234 23.088 6.819 1.00 23.13 O ATOM 1326 O HOH C 22 −6.694 29.679 −1.523 1.00 16.30 O ATOM 1327 O HOH C 23 −11.710 19.332 2.424 1.00 24.16 O ATOM 1328 O HOH C 24 −14.256 29.319 1.426 1.00 12.04 O ATOM 1329 O HOH C 25 3.237 23.322 −13.274 1.00 29.42 O ATOM 1330 O HOH C 26 20.289 18.807 −15.841 1.00 19.78 O ATOM 1331 O HOH C 27 −1.594 31.984 6.807 1.00 24.42 O ATOM 1332 O HOH C 28 3.075 24.510 −9.441 1.00 13.80 O ATOM 1333 O HOH C 29 0.606 22.808 −14.625 1.00 26.68 O ATOM 1334 O HOH C 30 26.106 25.904 6.889 1.00 24.57 O ATOM 1335 O HOH C 31 −4.094 27.131 4.823 1.00 19.04 O ATOM 1336 O HOH C 32 25.045 24.511 −3.504 1.00 21.00 O ATOM 1337 O HOH C 33 17.606 22.700 −13.727 1.00 23.61 O ATOM 1338 O HOH C 34 5.486 17.490 −14.358 1.00 19.98 O ATOM 1339 O HOH C 35 4.890 27.658 −9.655 1.00 13.93 O ATOM 1340 O HOH C 36 10.766 13.724 −11.495 1.00 18.42 O ATOM 1341 O HOH C 37 4.052 28.784 12.539 1.00 22.13 O ATOM 1342 O HOH C 38 1.576 29.602 8.266 1.00 18.99 O ATOM 1343 O HOH C 39 10.038 23.514 11.625 1.00 19.35 O ATOM 1344 O HOH C 40 1.589 27.268 12.084 1.00 25.47 O ATOM 1345 O HOH C 41 18.764 25.989 10.989 1.00 22.75 O ATOM 1346 O HOH C 42 3.139 31.954 10.647 1.00 19.30 O ATOM 1347 O HOH C 43 −0.698 23.830 −11.690 1.00 27.71 O ATOM 1348 O HOH C 44 7.324 34.542 −3.438 1.00 19.70 O ATOM 1349 O HOH C 45 15.393 25.076 −12.431 1.00 18.83 O ATOM 1350 O HOH C 46 −2.952 24.923 −10.653 1.00 20.01 O ATOM 1351 O HOH C 47 8.526 27.347 −11.572 1.00 14.42 O ATOM 1352 O HOH C 48 25.003 29.995 1.360 1.00 18.25 O ATOM 1353 O HOH C 49 8.851 4.740 −0.804 1.00 44.60 O ATOM 1354 O HOH C 50 2.150 15.238 10.244 1.00 23.34 O ATOM 1355 O HOH C 51 23.248 31.543 −8.292 1.00 38.43 O ATOM 1356 O HOH C 52 11.925 37.161 11.540 1.00 21.41 O ATOM 1357 O HOH C 53 7.506 26.580 12.158 1.00 17.84 O ATOM 1358 O HOH C 54 22.403 20.582 5.479 1.00 22.66 O ATOM 1359 O HOH C 55 2.920 4.070 −8.880 1.00 27.70 O ATOM 1360 O HOH C 56 13.365 20.446 −14.539 1.00 31.29 O ATOM 1361 O HOH C 57 3.205 6.214 5.047 1.00 28.77 O ATOM 1362 O HOH C 58 11.306 34.224 14.484 1.00 29.77 O ATOM 1363 O HOH C 59 13.968 10.048 1.479 1.00 18.69 O ATOM 1364 O HOH C 60 25.949 27.531 9.261 1.00 31.24 O ATOM 1365 O HOH C 61 17.403 29.407 −7.478 1.00 16.15 O ATOM 1366 O HOH C 62 4.988 4.691 −5.306 1.00 17.42 O ATOM 1367 O HOH C 63 0.885 22.565 −9.915 1.00 21.01 O ATOM 1368 O HOH C 64 13.472 22.874 −13.065 1.00 18.40 O ATOM 1369 O HOH C 65 7.728 21.661 13.603 1.00 23.40 O ATOM 1370 O HOH C 66 0.826 17.054 11.904 1.00 20.26 O ATOM 1371 O HOH C 67 22.082 14.632 −14.560 1.00 28.15 O ATOM 1372 O HOH C 68 −8.011 11.229 −10.833 1.00 28.48 O ATOM 1373 O HOH C 69 19.869 21.156 −14.466 1.00 28.81 O ATOM 1374 O HOH C 70 8.505 29.865 −10.308 1.00 18.39 O ATOM 1375 O HOH C 71 9.722 24.355 −14.345 1.00 22.96 O ATOM 1376 O HOH C 72 −9.383 9.208 −12.697 1.00 37.48 O ATOM 1377 O HOH C 73 13.683 12.690 −5.641 1.00 16.03 O ATOM 1378 O HOH C 74 22.703 16.963 4.759 1.00 25.06 O ATOM 1379 O HOH C 75 −4.661 12.064 5.165 1.00 23.98 O ATOM 1380 O HOH C 76 −0.116 11.262 4.335 1.00 15.82 O ATOM 1381 O HOH C 77 10.964 33.126 −7.385 1.00 22.22 O ATOM 1382 O HOH C 78 12.741 11.275 −3.598 1.00 26.61 O ATOM 1383 O HOH C 79 17.124 9.812 −1.247 1.00 24.04 O ATOM 1384 O HOH C 80 10.507 29.065 14.092 1.00 42.45 O ATOM 1385 O HOH C 81 1.932 30.283 −11.189 1.00 26.71 O ATOM 1386 O HOH C 82 6.515 32.337 −7.631 1.00 23.56 O ATOM 1387 O HOH C 83 5.835 14.658 10.607 1.00 26.21 O ATOM 1388 O HOH C 84 16.286 9.808 −5.465 1.00 32.46 O ATOM 1389 O HOH C 85 17.982 26.013 −14.610 1.00 41.22 O ATOM 1390 O HOH C 86 6.757 25.556 −14.889 1.00 32.09 O ATOM 1391 O HOH C 87 12.168 23.328 14.403 1.00 32.02 O ATOM 1392 O HOH C 88 25.612 18.037 0.846 1.00 37.22 O ATOM 1393 O HOH C 89 −11.612 11.941 −3.966 1.00 35.20 O ATOM 1394 O HOH C 90 16.837 22.905 14.860 1.00 45.41 O ATOM 1395 O HOH C 91 4.324 6.909 8.884 1.00 35.98 O ATOM 1396 O HOH C 92 −0.508 30.459 −7.690 1.00 31.56 O ATOM 1397 O HOH C 93 23.798 25.345 −12.973 1.00 40.97 O ATOM 1398 O HOH C 94 −1.156 31.732 −3.874 1.00 22.96 O ATOM 1399 O HOH C 95 24.773 23.540 7.683 1.00 31.69 O ATOM 1400 O HOH C 96 13.370 28.961 14.118 1.00 25.13 O ATOM 1401 O HOH C 97 11.176 32.694 −14.976 1.00 45.55 O ATOM 1402 O HOH C 98 3.247 33.923 −4.564 1.00 23.20 O ATOM 1403 O HOH C 99 0.655 1.887 −9.968 1.00 33.74 O ATOM 1404 O HOH C 100 −1.605 27.211 6.238 1.00 25.07 O ATOM 1405 O HOH C 101 27.031 15.167 −5.294 1.00 41.18 O ATOM 1406 O HOH C 102 12.701 37.927 −2.640 1.00 24.98 O ATOM 1407 O HOH C 103 11.242 17.624 16.856 1.00 28.15 O ATOM 1408 O HOH C 104 −0.714 21.375 15.259 1.00 26.94 O ATOM 1409 O HOH C 105 11.116 20.520 −15.920 1.00 32.85 O ATOM 1410 O HOH C 106 26.599 26.380 −4.765 1.00 27.41 O ATOM 1411 O HOH C 107 −3.531 30.025 −4.711 1.00 31.33 O ATOM 1412 O HOH C 108 −10.386 14.344 6.766 1.00 28.46 O ATOM 1413 O HOH C 109 −0.920 7.733 −14.875 1.00 34.53 O ATOM 1414 O HOH C 110 3.075 25.899 −13.882 1.00 43.70 O ATOM 1415 O HOH C 111 3.494 13.321 11.851 1.00 41.80 O ATOM 1416 O HOH C 112 25.115 15.319 −15.164 1.00 51.77 O ATOM 1417 O HOH C 113 −10.814 3.486 −8.023 1.00 53.15 O ATOM 1418 O HOH C 114 −7.002 14.595 3.801 1.00 22.94 O ATOM 1419 O HOH C 115 22.295 23.908 11.184 1.00 37.30 O ATOM 1420 O HOH C 116 10.413 13.556 −14.122 1.00 31.05 O ATOM 1421 O HOH C 117 18.144 8.726 1.052 1.00 25.56 O ATOM 1422 O HOH C 118 8.111 12.858 17.399 1.00 26.70 O ATOM 1423 O HOH C 119 −1.900 6.427 −5.476 1.00 37.96 O ATOM 1424 O HOH C 120 26.236 17.529 −6.312 1.00 42.03 O ATOM 1425 O HOH C 121 14.867 33.188 −10.053 1.00 29.76 O ATOM 1426 O HOH C 122 18.107 30.034 −14.340 1.00 29.39 O ATOM 1427 O HOH C 123 26.086 20.699 0.543 1.00 39.98 O ATOM 1428 O HOH C 124 21.347 13.146 4.294 1.00 42.70 O ATOM 1429 O HOH C 125 −5.395 27.500 −5.225 1.00 32.34 O ATOM 1430 O HOH C 126 19.591 11.720 −11.515 1.00 26.26 O ATOM 1431 O HOH C 127 −6.132 8.228 1.360 1.00 31.16 O ATOM 1432 O HOH C 128 5.359 23.142 −15.118 1.00 29.53 O ATOM 1433 O HOH C 129 28.708 29.233 −0.696 1.00 46.73 O ATOM 1434 O HOH C 130 10.613 5.449 6.251 1.00 28.10 O ATOM 1435 O HOH C 131 −0.007 6.005 −1.297 1.00 32.11 O ATOM 1436 O HOH C 132 23.368 15.050 11.673 1.00 37.46 O ATOM 1437 O HOH C 133 25.046 19.722 −8.120 1.00 33.05 O ATOM 1438 O HOH C 134 9.424 25.141 13.565 1.00 33.26 O ATOM 1439 O HOH C 135 25.847 14.252 11.288 1.00 34.74 O ATOM 1440 O HOH C 136 24.817 29.704 8.899 1.00 32.97 O ATOM 1441 O HOH C 137 24.296 33.831 12.484 1.00 33.85 O ATOM 1442 O HOH C 138 8.700 27.899 −14.193 1.00 28.62 O ATOM 1443 O HOH C 139 23.158 11.855 −6.761 1.00 44.27 O ATOM 1444 O HOH C 140 5.534 11.801 10.878 1.00 29.38 O ATOM 1445 O HOH C 141 −5.645 31.000 −3.651 1.00 30.23 O ATOM 1446 O HOH C 142 15.884 18.585 15.441 1.00 38.52 O ATOM 1447 O HOH C 143 1.856 16.886 14.584 1.00 31.21 O ATOM 1448 O HOH C 144 29.245 25.474 −4.694 1.00 42.40 O ATOM 1449 O HOH C 145 −8.458 20.496 −7.555 1.00 33.58 O ATOM 1450 O HOH C 146 −0.833 29.488 7.418 1.00 24.32 O ATOM 1451 O HOH C 147 5.247 30.037 −11.175 1.00 35.52 O ATOM 1452 O HOH C 148 9.483 21.937 −14.176 1.00 30.20 O ATOM 1453 O HOH C 149 15.295 20.153 18.089 1.00 48.34 O ATOM 1454 O HOH C 150 0.542 30.543 10.777 1.00 38.34 O ATOM 1455 O HOH C 151 9.387 2.527 3.565 1.00 30.25 O ATOM 1456 O HOH C 152 18.283 19.624 14.935 1.00 31.09 O ATOM 1457 O HOH C 153 2.231 32.712 −10.463 1.00 37.34 O ATOM 1458 O HOH C 154 3.494 26.236 15.976 1.00 35.76 O ATOM 1459 O HOH C 155 0.000 0.000 −3.140 0.33 36.16 O ATOM 1460 O HOH C 156 29.599 26.684 4.137 1.00 49.58 O ATOM 1461 O HOH C 157 20.265 30.811 −13.355 1.00 32.27 O ATOM 1462 O HOH C 158 4.118 21.799 15.115 1.00 43.85 O ATOM 1463 O HOH C 159 −1.282 15.362 11.929 1.00 38.31 O ATOM 1464 O HOH C 160 7.000 32.634 14.785 1.00 58.41 O ATOM 1465 O HOH C 161 −11.662 18.007 −11.387 1.00 37.98 O ATOM 1466 O HOH C 162 8.620 38.125 12.742 1.00 41.89 O ATOM 1467 O HOH C 163 0.755 6.442 8.564 1.00 37.57 O ATOM 1468 O HOH C 164 9.657 19.932 14.077 1.00 33.41 O ATOM 1469 O HOH C 165 20.648 32.613 −5.684 1.00 34.32 O ATOM 1470 O HOH C 166 −2.891 4.747 −3.845 1.00 36.48 O ATOM 1471 O HOH C 167 0.599 8.421 11.145 1.00 51.34 O ATOM 1472 O HOH C 168 24.255 8.505 6.368 1.00 58.21 O ATOM 1473 O HOH C 169 4.598 7.586 11.519 1.00 51.50 O ATOM 1474 O HOH C 170 6.277 12.729 −15.260 1.00 36.41 O ATOM 1475 O HOH C 171 2.242 33.629 −6.905 1.00 36.85 O ATOM 1476 O HOH C 172 −4.826 29.572 5.929 1.00 25.80 O ATOM 1477 O HOH C 173 2.215 34.911 0.535 1.00 31.20 O ATOM 1478 O HOH C 174 18.906 31.815 −7.966 1.00 26.96 O ATOM 1479 O HOH C 175 5.859 36.246 −1.608 1.00 19.07 O ATOM 1480 O HOH C 176 27.506 12.770 9.118 1.00 36.92 O ATOM 1481 O HOH C 177 −2.231 5.718 7.807 1.00 31.70 O ATOM 1482 O HOH C 178 −9.593 14.887 4.372 1.00 32.26 O ATOM 1483 O HOH C 179 24.429 26.258 11.604 1.00 36.78 O ATOM 1484 O HOH C 180 26.276 10.158 6.989 1.00 40.95 O ATOM 1485 O HOH C 181 15.659 31.633 −14.927 1.00 33.21 O ATOM 1486 O HOH C 182 6.439 5.851 12.056 1.00 40.31 O ATOM 1487 O HOH C 183 23.338 20.398 14.296 1.00 46.16 O ATOM 1488 O HOH C 184 −7.590 15.805 −11.158 1.00 30.12 O ATOM 1489 O HOH C 185 25.223 36.478 −5.843 1.00 51.46 O ATOM 1490 O HOH C 186 14.494 32.637 −12.694 1.00 38.92 O ATOM 1491 O HOH C 187 −1.688 32.074 11.590 1.00 43.26 O ATOM 1492 O HOH C 188 9.145 35.161 −11.070 1.00 42.52 O ATOM 1493 O HOH C 189 25.406 16.009 8.614 1.00 54.32 O ATOM 1494 O HOH C 190 23.108 17.782 14.398 1.00 49.04 O ATOM 1495 O HOH C 191 4.797 35.701 2.113 1.00 13.86 O ATOM 1496 O HOH C 192 11.313 8.379 9.732 1.00 14.54 O ATOM 1497 O HOH C 193 25.138 8.254 9.504 1.00 40.56 O ATOM 1498 O HOH C 194 −5.248 31.572 4.359 1.00 16.20 O ATOM 1499 O HOH C 195 3.552 34.777 −1.836 1.00 16.79 O ATOM 1500 O HOH C 196 15.843 31.625 −0.658 1.00 14.25 O ATOM 1501 O HOH C 197 18.260 37.087 9.650 1.00 29.40 O ATOM 1502 O HOH C 198 6.016 16.515 17.366 1.00 32.02 O ATOM 1503 O HOH C 199 18.173 32.351 1.662 1.00 16.19 O ATOM 1504 O HOH C 200 −6.339 1.106 −9.354 1.00 30.70 O ATOM 1505 O HOH C 201 9.356 2.408 −1.635 1.00 41.48 O ATOM 1506 O HOH C 202 −2.957 29.499 −7.472 1.00 39.78 O ATOM 1507 O HOH C 203 16.718 37.857 −3.857 1.00 53.51 O ATOM 1508 O HOH C 204 24.450 17.663 −9.932 1.00 43.97 O ATOM 1509 O HOH C 205 20.771 23.401 9.091 1.00 21.18 O ATOM 1510 O HOH C 206 17.991 14.996 15.334 1.00 34.03 O ATOM 1511 O HOH C 207 14.785 13.418 15.035 1.00 23.11 O ATOM 1512 O HOH C 208 21.364 10.070 0.938 1.00 43.98 O ATOM 1513 O HOH C 209 17.446 32.692 15.044 1.00 32.28 O ATOM 1514 O HOH C 210 −4.097 −0.566 −2.525 1.00 43.94 O ATOM 1515 O HOH C 211 −7.601 16.797 −13.642 1.00 30.53 O ATOM 1516 O HOH C 212 −6.402 17.493 −9.417 1.00 32.47 O ATOM 1517 O HOH C 213 20.479 12.017 −6.368 1.00 41.20 O ATOM 1518 O HOH C 214 26.233 15.387 −8.763 1.00 61.51 O ATOM 1519 O HOH C 215 8.171 30.282 14.273 1.00 47.43 O ATOM 1520 O HOH C 216 −4.860 2.481 −3.365 1.00 36.97 O ATOM 1521 O HOH C 217 7.253 13.009 −17.778 1.00 34.50 O ATOM 1522 O HOH C 218 31.889 26.105 0.328 1.00 48.69 O ATOM 1523 O HOH C 219 23.801 31.899 −5.289 1.00 37.45 O ATOM 1524 O HOH C 220 8.049 20.377 15.946 1.00 46.50 O ATOM 1525 O HOH C 221 1.128 35.425 −1.061 1.00 32.18 O ATOM 1526 O HOH C 222 0.736 3.120 −12.959 1.00 49.39 O ATOM 1527 O HOH C 223 5.714 34.194 −5.598 1.00 45.92 O ATOM 1528 O HOH C 224 32.462 23.398 −0.604 1.00 52.94 O ATOM 1529 O HOH C 225 22.920 12.668 −2.396 1.00 42.31 O ATOM 1530 O HOH C 226 −10.468 20.039 −4.505 1.00 31.70 O ATOM 1531 O HOH C 227 −9.571 20.524 7.691 1.00 33.23 O ATOM 1532 O HOH C 228 −5.512 24.361 −11.421 1.00 41.21 O ATOM 1533 O HOH C 229 29.130 19.154 −2.128 1.00 44.72 O ATOM 1534 O HOH C 230 26.079 16.595 −13.126 1.00 40.17 O ATOM 1535 O HOH C 231 8.249 33.990 −6.344 1.00 37.22 O ATOM 1536 O HOH C 232 −6.369 23.852 −2.542 1.00 30.86 O ATOM 1537 O HOH C 233 28.025 13.978 −14.597 1.00 41.20 O ATOM 1538 O HOH C 234 4.526 27.966 −13.975 1.00 57.38 O ATOM 1539 O HOH C 235 19.662 19.264 17.253 1.00 45.54 O ATOM 1540 O HOH C 236 6.207 28.046 13.913 1.00 39.03 O ATOM 1541 O HOH C 237 21.469 21.107 19.172 1.00 36.45 O ATOM 1542 O HOH C 238 20.241 3.932 14.047 1.00 24.86 O ATOM 1543 O HOH C 239 23.003 12.985 −9.593 1.00 41.28 O ATOM 1544 O HOH C 240 20.820 20.952 10.196 1.00 26.99 O ATOM 1545 O HOH C 241 −13.043 10.647 −0.957 1.00 44.71 O ATOM 1546 O HOH C 242 −12.463 23.019 9.434 1.00 35.64 O ATOM 1547 O HOH C 243 16.937 13.054 16.827 1.00 28.17 O ATOM 1548 O HOH C 244 10.816 36.185 −5.880 1.00 34.97 O ATOM 1549 O HOH C 245 29.507 38.709 0.897 1.00 30.98 O ATOM 1550 O HOH C 246 −6.984 19.997 −9.497 1.00 36.75 O ATOM 1551 O HOH C 247 23.227 8.607 1.927 1.00 36.74 O ATOM 1552 O HOH C 248 7.431 1.768 −12.583 1.00 34.07 O ATOM 1553 O HOH C 249 −1.145 35.666 −3.730 1.00 32.73 O ATOM 1554 O HOH C 250 −10.303 18.635 9.413 1.00 27.27 O ATOM 1555 O HOH C 251 24.497 11.680 −11.761 1.00 43.83 O ATOM 1556 O HOH C 252 22.035 −1.582 4.357 1.00 31.60 O ATOM 1557 O HOH C 253 12.859 34.955 −6.865 1.00 46.12 O ATOM 1558 O HOH C 254 −0.415 19.213 17.144 1.00 33.59 O ATOM 1559 O HOH C 255 −7.392 −0.762 −13.446 1.00 38.50 O ATOM 1560 O HOH C 256 22.357 24.323 19.921 1.00 39.95 O ATOM 1561 O HOH C 257 25.108 17.891 18.040 1.00 46.55 O ATOM 1562 O HOH C 258 −10.544 16.883 7.534 1.00 42.60 O ATOM 1563 O HOH C 259 −1.823 9.270 10.440 1.00 42.68 O ATOM 1564 O HOH C 260 0.038 32.968 −8.263 1.00 49.96 O ATOM 1565 O HOH C 261 −2.957 37.760 −3.649 1.00 45.46 O ATOM 1566 O HOH C 262 16.070 16.430 19.660 1.00 46.71 O ATOM 1567 O HOH C 263 −7.829 21.465 −12.380 1.00 37.90 O ATOM 1568 O HOH C 264 −5.381 10.775 10.912 1.00 54.26 O ATOM 1569 O HOH C 265 31.411 19.412 −9.999 1.00 57.72 O ATOM 1570 O HOH C 266 17.650 −0.220 11.442 1.00 25.86 O ATOM 1571 O HOH C 267 22.703 6.018 2.267 1.00 31.89 O ATOM 1572 O HOH C 268 24.287 −0.928 2.476 1.00 56.27 O ATOM 1573 O HOH C 269 0.000 0.000 5.917 0.33 41.08 O ATOM 1574 O HOH C 270 4.315 1.035 −18.133 1.00 46.45 O ATOM 1575 O HOH C 271 25.962 8.475 2.448 1.00 31.32 O ATOM 1576 O HOH C 272 22.171 26.495 18.447 1.00 33.86 O ATOM 1577 O HOH C 273 36.940 19.863 −1.222 1.00 38.39 O ATOM 1578 O HOH C 274 37.580 19.529 1.676 1.00 40.36 O ATOM 1579 O HOH C 275 −1.797 34.546 −7.351 1.00 42.77 O ATOM 1580 O HOH C 276 −13.663 15.413 7.393 1.00 33.48 O ATOM 1581 O HOH C 277 15.698 23.450 20.973 1.00 42.32 O ATOM 1582 O HOH C 278 −3.885 33.689 −6.005 1.00 52.43 O ATOM 1583 O HOH C 279 28.337 40.591 7.898 1.00 40.16 O ATOM 1584 O HOH C 280 33.930 23.410 −9.468 1.00 44.18 O ATOM 1585 O HOH C 281 2.806 9.012 12.959 1.00 47.36 O ATOM 1586 O HOH C 282 10.315 39.457 14.714 1.00 40.30 O ATOM 1587 O HOH C 283 −0.003 38.067 −8.793 0.33 46.57 O ATOM 1588 O HOH C 284 −0.827 9.206 6.958 1.00 24.57 O ATOM 1589 O HOH C 285 10.557 8.220 −2.795 1.00 29.67 O ATOM 1590 O HOH C 286 10.671 9.390 0.780 1.00 18.38 O ATOM 1591 O HOH C 287 2.511 37.077 1.303 1.00 27.43 O ATOM 1592 O HOH C 288 10.671 5.913 10.408 1.00 28.27 O ATOM 1593 O HOH C 289 12.179 9.861 −1.440 1.00 34.08 O ATOM 1594 O HOH C 290 9.048 7.318 −0.271 1.00 25.03 O ATOM 1595 O HOH C 291 −0.001 38.072 2.437 0.33 9.24 O

Example 6 Confirmation of Binding Abilities of Various Mutant Proteins

An interaction was confirmed by the method described in Example 1 except that mutant Hd3a and mutant GF14c were used. Mutant Hd3a (R55A, M63A, R64A, P96L, F103A, R132A, and R168A) and mutant GF14c (F200A, I204A, E212A, Y215A, and R226A) were produced using a Quickchange site-directed mutagenesis kit (Stratagene) according to the protocol of the kit. It should be noted that “R55A” means a mutant Hd3a protein having a substitution of arginine (R) at position 55 to alanine (A) in Hd3a.

FIG. 5 show the results. Out of the Hd3a mutants, M63A, R64A, P96L, F103A, and R132A each exhibited a reduction in binding ability to GF14c. In particular, F103A exhibited a remarkable reduction in binding ability (FIG. 5 a). Out of the GF14c mutants, F200A, I204A, E212A, and Y215A each exhibited a reduction in binding ability to Hd3a. In particular, F200A and I204A each exhibited a remarkable reduction in binding ability (FIG. 5 b).

Example 7 Gel-Shift Assay of Florigen Activation Complex and C-Box DNA

An arabidopsis AP1 promoter-derived sequence (22 base pairs: 5′-CTTCACGAGACGTCGATAATCA-3′ (SEQ ID NO: 5)) was used as C-box DNA. C-box DNA used for the assay was prepared by chemical synthesis. A 100 mM Tris-borate buffer containing 0.2 mM EDTA, 1.5 mM MgCl₂, and 5% glycerol was used for the preparation of a florigen activation complex. A conjugate of the florigen activation complex (FD1-GF14-Hd3a) (sometimes referred to as “FAC”) and C-box DNA was generated by mixing 30 pmol of C-box DNA, 40 pmol of OsFD1, 40 pmol of GF14c, and 80 pmol of Hd3a with each other and subjecting the mixture to a reaction through incubation at 4° C. for 30 minutes. The generated conjugate was subjected to electrophoresis using a 10% acrylamide gel and detected by ethidium bromide staining.

FIG. 6 shows the results. The florigen activation complex was found to bind to C-box DNA and form a stable complex on the DNA (lane 4). Hd3a is considered to form a florigen activation complex on a promoter and be responsible for the transcriptional activation of a gene necessary for flowering.

Example 8 Confirmation of Interactions Between Florigen and 14-3-3 Protein Isoforms

Interactions between respective proteins were confirmed using a yeast two-hybrid assay.

First, a plasmid was constructed as described below. Full-length cDNAs for OsGF14a (Os08g0480800), OsGF14b (Os04g0462500), OsGF14c (Os08g0430500), OsGF14d (Os11g0546900), OsGF14e (Os02g0580300), OsGF14f (Os03g0710800), OsGF14g (Os01g0209200), OsGF14h (Os11g0609600), and OsFD1 (Os09g0540800) were cloned by RT-PCR. The forward and reverse primers were designed based on genetic information in the rice DNA database. The full-length coding region was PCR-amplified and introduced into a pENTR/D-TOPO cloning vector (Invitrogen) to obtain entry clones.

The screening of Hd3a interactors was performed by the method described in Non Patent Literature 2. Yeast two-hybrid libraries were produced using total RNA extracted from wild-type leaf blades. After cDNA synthesis using a cDNA synthesis kit (Stratagene), the cDNAs were inserted into a pVP16 vector (Hollenberg et al., 1995) and introduced into a yeast L40 strain. As a bait, the full-length Hd3a ORF was cloned into a pBTM116 vector (Bartel et al., 1993). Screening was performed on an SC medium lacking histidine and containing 2.5 mM 3-aminotriazole (3-AT).

For an interaction assay, pBTM116 and pVP16 were converted to pBTM116-GW and pVP16-GW, respectively, using a Gateway vector conversion system (Invitrogen) according to the manufacturer's instructions. Yeast cells were grown at 30° C. for 5 days using an SC medium (without uracil, tryptophan, leucine, and histidine), the medium containing added histidine or 1 to 10 mM 3-aminotriazole (+His or +3-AT). The concentration of 3-AT was determined by a bait-prey combination.

FIG. 7 shows the results. GF14b, c, e, and f (OsGF14b, c, e, and f) were each found to be exhibit an interaction with Hd3a. GF14b was used in the following examples.

Example 9 Confirmation of Sites Contributing to Interactions Among Three Proteins

Various mutant proteins were produced by constructing plasmids as described below. The introduction of amino-acid substitutions or deletions into OsGF14, OsFD1, and Hd3a was performed by PCR with KOD FX DNA polymerase (TOYOBO). The PCR-amplified fragments were cloned into a pENTR/D-TOPO cloning vector (Invitrogen) to obtain entry clones. Mutant proteins expressed in the obtained entry clones are as follows: S192A and S192E for OsFD1; R64A/R68A, I277A/L230A, F206A, I210A, E212A, E218A, Y221A, R232A, and D233A for OsGF14b; and R64G, R64A, R64K, T68I, P96L, F103A, R132A, R132K, R64G/R132A, and R64K/R132K for Hd3a. Interactions were confirmed in the same manner as in Example 8 using those entry clones.

It should be noted that, out of the mutant proteins, a protein indicated using “/” is a multiple mutant protein, and for example, “R64G/R132A” for Hd3a means a double-mutant Hd3a protein having a substitution of arginine (R) at position 64 to glycine (G) and a substitution of arginine (R) at position 132 to alanine (A).

FIGS. 8 a to 8 d show the results.

The phosphorylation of serine at position 192 in OsFD1 was found to be important for an interaction between OsFD1 and OsGF14b (FIG. 8 a).

Out of the mutant proteins for OsGF14b, in the mutant proteins having substitutions in F206, I210, E212, E218, Y221, R232, and D233, there were reductions in binding ability to Hd3a, whereas there was no influence on binding ability to OsFD1 (FIG. 8 c).

Out of the mutant proteins for Hd3a, in the mutant proteins having substitutions in R64, T68, P96, F103, and 8132, there were remarkable reductions in binding ability to OsGF14b (FIG. 8 d). Out of the mutant proteins for Hd3a, in the cases of P77L and R121H, there was no influence on binding ability to OsGF14b, suggesting that those sites were not important for binding to the 14-3-3 protein.

Example 10 Confirmation of Interaction in Shoot Apex of Rice

An interaction in the shoot apex of a rice plant was confirmed using an in vivo pull-down assay.

(1) Used Plant Materials

Rice (Oryza sativa L. subsp. Japonica) variety Norin 8 was used as a wild-type. Transgenic rice (pHd3a::Hd3a::GFP, p35S::GFP, and prolC::Hd3a::GFP) were produced as described in Tamaki et al., Science 316, 1033 (2007) and Okano et al., Plant J. 53, 65 (2008). The transgenic rice was produced using Agrobacterium-mediated transformation of rice calli, as described in Hiei et al. Plant J. 6, 271 (1994), and a hygromycin-resistant plant was produced from the transformed calli. Transgene integration was further confirmed by PCR amplification of a hygromycin phosphotransferase (HPT) gene in genome DNA extracted from the produced plant. It should be noted that pHd3a::Hd3a::GFP refers to transgenic rice having introduced therein a gene in which a promoter region in Hd3a, a full-length coding region in Hd3a, and a coding region in GFP are conjugated to each other, and is hereinafter sometimes abbreviated as “HHG.” p35S::GFP refers to transgenic rice having introduced therein a gene in which a CaMV 35S promoter and a coding region in GFP are conjugated to each other, and is hereinafter sometimes abbreviated as “35S GFP.” prolC::Hd3a::GFP refers to transgenic rice having introduced therein a gene in which a promoter region in rolC, a full-length coding region in Hd3a, and a coding region in GFP are conjugated to each other.

(3) In Vivo Pull-Down Assay

Samples of the shoot apical site were collected by microscopic dissection from HHG transgenic rice and 35S GFP transgenic rice. After grinding of the samples, the powder of each of the samples was dissolved in 100 μl of an extraction buffer (150 mM NaCl, 50 mM Tris, 0.1% Tween-20, 10% glycerol, 1 mM DTT, 1 mM Pefabloc SC (Roche), 1× Complete Proteinase Inhibitor Cocktail (Roche), and 1× Halt Phosphatase Inhibitor Cocktail (Pierce)), followed by mixing. After centrifugation (15 min, 4° C., 15,000 rcf), the supernatant was transferred to a tube, and the amount of a protein was measured by a Coomassie staining assay. An equivalent to the total amount of each of the proteins extracted from HHG transgenic rice and 35S GFP transgenic rice was incubated with 50 μl of anti-GFP MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions except that the following modifications was made: a flow-through was collected and used for further analysis. The column was rinsed four times with 200 μl of an extraction buffer (150 mM NaCl, 50 mM Tris, 0.1% Tween-20, 10% glycerol, 1 mM DTT, 1 mM Pefabloc SC (Roche), 1× Complete Proteinase Inhibitor Cocktail (Roche), and 1× Halt Phosphatase Inhibitor Cocktail (Pierce)). After that, the column was washed once with 100 μl of a low ionic buffer (20 mM TrisHCl, pH 7.5). After that, the column was removed from the magnetic field, and the remaining protein was extracted with 50 μl of a lysis buffer. The eluate was separated by 12.5% SDS-PAGE and subjected to immunoblotting using a primary antibody (polyclonal rabbit anti-GFP antibody (Abcam)) and an anti-14-3-3 antibody (provided by Dr. Yohsuke Takahashi (Hiroshima University, Japan)). After washing with TBST, the membrane was incubated for 1 hour with anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare). Detection was performed using enhanced chemiluminescence (ECL) protein gel blot detection reagents (GE Healthcare), and visualization was performed using an LAS-4000 mini Imager (Fujifilm).

FIG. 9 shows the results. The left side (input) of FIG. 9 shows the results in the case of extracting a protein from shoot apex cells of transgenic rice expressing GFP or an Hd3a-GFP-fused protein (35S GFP transgenic rice or HHG transgenic rice) and subjecting the extract to immunoblotting using an anti-GFP antibody (α-GFP) and an anti-14-3-3 antibody (α-14-3-3). The right side (IP-GFP) of FIG. 9 shows the results in the case of subjecting an extract obtained by extracting a protein from shoot apex cells to immunoprecipitation (IP) with an anti-GFP antibody (α-GFP), followed by immunoblotting in the same manner as described above. When the Hd3a-GFP-fused protein was expressed (HHG extract), a 14-3-3 protein was observed during co-precipitation with the anti-GFP antibody. On the other hand, when GFP was expressed alone (35S GFP extract), a 14-3-3 protein was not observed during co-precipitation. This revealed that Hd3a interacted with the 14-3-3 protein in the shoot apex of rice.

Example 11 Subcellular Localization of Three Proteins and Complex Thereof

Subcellular localization and bimolecular fluorescence complementation (BiFC method) were performed as described below.

First, Hd3a, GF14b, OsFD1, and β-glucuronidase (GUS) coding regions were cloned into fluorescent protein expression vectors or BiFC vectors and purified using a Purelink Plasmid Midiprep Kit (Invitrogen). For Hd3a, GF14b, OsFD1, and mutant proteins thereof, full-length coding regions were used. A vector expressing GFP, CFP, or mCherry was used as each of the fluorescent protein expression vectors. A vector expressing a Vn or Vc tag was used as each of the BiFC vectors. Vn or Vc emits fluoresce as Venus (mVenus) when exists in proximity.

(1) The transformation of rice Oc protoplasts was performed by a method described in Kyozuka and Shimamoto 1991, Plant J. 6, 271 (1994), Non Patent Literature 1, or the like. Fluorescent protein expression vectors having introduced therein various proteins were introduced into a protoplast suspension (2×10⁷ protoplasts/ml) by a PEG method (PEG-mediated method). After incubation at 30° C. for 24 hours, transformed protoplasts were obtained and used for microscopic observation.

In a co-expression system of two kinds of proteins, 1 μg of a GFP-GF14b expression plasmid or an Hd3a-mCherry expression plasmid and 10 μg of an NLS-CFP, CFP-OsFD1, or CFP-OsFD1 S192A expression plasmid were co-transformed into protoplasts. NLS refers to a nuclear localization signal peptide.

FIG. 10 shows the results. Fluorescence from Hd3a-mCherry was detected in both the cytoplasm and the nucleus, fluorescence from GFP-GF14b was detected in the cytoplasm, and fluorescence from CFP-OsFD1 was detected in the nucleus.

(2) In the same manner as in the method of (1), 5 μg of a Vn-fused protein expression vector and a Vc-fused protein expression vector were co-transformed in the BiFC method.

An mCherry expression plasmid was introduced simultaneously as a marker for transformation efficiency.

In order to quantify protein-protein interactions, the fluorescence intensities of mCherry and Venus from about 20 cells of various protoplasts having introduced therein expression plasmids for BiFC analysis (a Vn-fused protein expression vector, a Vc-fused protein expression vector, and an mCherry expression plasmid) were measured under the same microscope settings, and a Venus/mCherry value was calculated. In the experimental settings, a BiFC signal in cells showing Venus/mCherry values of >0.33 for an interaction between Hd3a and GF14b and Venus/mCherry values >0.4 for an interaction between GF14 band OsFD1 and an interaction between Hd3a and OsFD1 was recognized as reliable one. Therefore, the number of cells showing higher values than the above-mentioned values was scored.

FIG. 11 shows the results.

Vn or Vc is conjugated to the N-terminal and the C-terminal of Hd3a and GF14b and expressed in rice protoplasts. As a result, BiFC signals were confirmed in the cytoplasm for the interaction between Hd3a and GF14b, which were dependent on mutations in R64 and R132 of Hd3a (FIG. 11 a). In the BiFC method, mutations in R58 and R62 of GF14b did not affect the interaction between Hd3a and GF14b.

Next, an interaction between GF14b and OsFD1 was confirmed. As a result, the interaction therebetween was not observed in the cytoplasm. The interaction between GF14b and OsFD1 was detected mainly in the nucleus, and only the localization of GF14b was confirmed in the cytoplasm (FIG. 11 b). A mutation in 5192 of OsFD1 was found to inhibit the interaction with GF14b. Further, in the BiFC method, mutations in R58 and R132 of GF14b did not affect the interaction with OsFD1.

An interaction between Hd3a and OsFD1 was confirmed. As a result, a complex of Hd3a and OsFD1 was found to be present in the nucleus (FIG. 11 c). Mutations in R64 and R132 of Hd3a or a mutation in 5192 of OsFD1 inhibited the interaction between Hd3a and OsFD1.

(3) In the same manner as in the method of (1), cells co-expressing OsFD1 in a BiFC system for confirming an interaction between Hd3a and GF14b were produced, and the amount of nuclear accumulation of the BiFC signal was examined. In order to co-express NLS-CFP and CFP-OsFD1 in the BiFC method, 10 μg of an NLS-CFP expression plasmid and a CFP-OsFD1 expression plasmid were used.

A method of calculating the amount of nuclear accumulation was established. First, the fluorescence intensities of Venus and mCherry in the nuclei of transformed cells were measured. After that, a value of (Venus in nucleus/mCherry in nucleus)/(Venus in whole cell/mCherry in whole cell) was calculated. This value indicates a ratio between the amounts of nuclear accumulation of Venus and mCherry. Those values and the corresponding confocal images obtained from each cell were compared to each other. As compared to mCherry distribution, values of more than 1.2 were recognized as nuclear localization, values of 0.8 to 1.2 were recognized as localization in both the nucleus and the cytoplasm, and values of less than 0.8 were recognized as cytoplasmic localization.

FIG. 12 shows the results.

In the case of co-expression of GFP-GF14b with NLS-CFP, fluorescence from GFP was confirmed in the cytoplasm. In the case of transient co-expression of GFP-GF14b with CFP-OsFD1, fluorescence from CFP was confirmed in the nucleus, and fluorescence from GFP was also observed in the nucleus, which remarkably differed from the case of the co-expression with NLS-CFP. On the other hand, in the case of co-expression of an OsFD1 mutant protein S192A with GFP-GF14b, the movement of fluorescence from GFP into the nucleus was not observed (FIG. 12 a). This suggested that the localization of GF14b was transferred from the nucleus to the cytoplasm owing to the presence of OsFD1.

In the case of co-expression of Hd3a-mCherry with CFP-OsFD1, the amount of nuclear accumulation of mCherry increased as compared to the case of the co-expression with CFP alone. In the case of co-expression of an OsFD1 mutant protein S192A with Hd3a-mCherry, no increase in the amount of nuclear accumulation was found (FIG. 12 b). Those results suggest that Hd3a and GF14b move from the cytoplasm to the nucleus to form a complex with OsFD1.

In order to confirm the influence of OsFD1 on the localization of a complex of Hd3a and GF14b, the BiFC method and CFP-OsFD1 were co-expressed. When OsFD1 was not expressed, the complex of Hd3a and GF14b (BiFC signal) was found to be present in the cytoplasm. In addition, when CFP-OsFD1 is co-expressed, the complex of Hd3a and GF14b was clearly observed predominantly in the nucleus (FIG. 12 c). The movement of the complex of Hd3a and GF14b by OsFD1 co-expression was reduced by introducing mutations R58A/R62A into GF14b. For the localization of each protein in rice cells, it was suggested that the complex of Hd3a and GF14b was first formed in the cytoplasm, and the complex of Hd3a and GF14b interacted with OsFD1 in the nucleus through an interaction between GF14b and OsFD1.

Example 12 Influence of Interaction Among OsFD1, GF14b, and Hd3a on OsMADS15 Transcriptional Control

The influence of interactions among OsFD1, GF14b, and Hd3a on OsMADS15 transcriptional control was confirmed using a transient expression assay using protoplasts.

The transformation of rice Oc protoplasts was performed by a method described in Kyozuka and Shimamoto 1991, Plant J. 6, 271 (1994), Non Patent Literature 1, or the like. 8 μg of Hd3a expression vectors and 16 μg of OsFD1 expression vectors were introduced into 500 μl of a protoplast suspension (2×10⁷ protoplasts/ml) by a PEG method (PEG-mediated method). After incubation at 30° C. for 24 hours, the protoplast suspension was centrifuged and the cell pellet was frozen at −80° C. for RNA extraction.

pGII pUbq GW-T7 and pGII pUbq HA-GW were produced by inserting a ubiquitin promoter derived from maize, an NOS termination region (Miki and Shimamoto, Plant Cell Physiol. 45, 490 (2004)), and an attR recombination region with a T7 tag region or an HA tag region (Nakagawa et al., 2007) into a pGreen II vector (Hellens et al., Plant Mol. Biol. 42, 819 (2000)).

An Hd3a gene, an OsFD1 gene, or a mutant gene thereof incorporated in a pENTR D-TOPO vector was transformed into pGII pUbq GW-T7 and pGII pUbq HA-GW by a Gateway recombination method with Gateway BP clonase II (Invitrogen) (pGIIpUbqHd3a-T7, pGIIpUbqHA-OsFD1, pGIIpUbqHA-OsFD1, and pGIIpUbqHd3a-T7). Various Hd3a expression vectors and OsFD1 expression vectors were introduced into protoplasts to express various proteins, and the RNA expression amount of OsMADS15 was confirmed. It should be noted that OsGF14 is generally constantly expressed in all cells, and hence it is considered to be impossible to confirm its effect even when introduced.

(2) RNA extraction and real-time PCR analysis were performed as described below.

Leaf blades of wild-type and transgenic plants were harvested. Total RNA was extracted using a TRizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized from 1 or 2 μg of RNA using an oligo dT primer (21-mer) and reverse-transcribed with Superscript II reverse transcriptase (Invitrogen). 1 μl of cDNA was used for quantitative analysis of gene expression using SYBR Green PCR master mix (Applied Biosystems) with gene-specific primers. Data was collected using an ABI PRISM 7000 sequence detection system according to the manual. Primers for ubiquitin and OsMADS15 are as described in Komiya et al., Development 135, 767 (2008). Primers for GF14b and GF14e are as follows:

GF14b-realF (SEQ ID NO: 4) GCGGAAGAGATCAGGGAAG; GF14b realR (SEQ ID NO: 5) CGACAACAATCACAGCCACA; GF14e realF (SEQ ID NO: 6) CAAGGGCGAATCAGGAGA; and GF14e realR (SEQ ID NO: 7) TGAAACGATAACCCACAGCA.

FIG. 13 shows the results.

The amount of OsMADS15 RNA increased with time after Hd3a and OsFD1 co-transformation. Hd3a or OsFD1 alone did not increase the RNA expression amount of OsMADS15. The OsMADS15 activation was found to start 4 hours after transformation, suggesting quick responsiveness between Hd3a and OsFD1 (FIG. 13 a).

FIG. 13 b shows the results of co-expression of various Hd3a mutants with OsFD1. Mutant Hd3a Y87H did not promote OsMADS15 transcription. Mutant Hd3a R64G and R132A slightly reduced OsMADS15 expression. Mutant Hd3a R64K and R64K/R132K remarkably reduced OsMADS15 expression. Those results suggest that the interaction between Hd3a and OsGF14 is required for OsMADS15 expression, and hydrophobicity, not a positive charge, of arginine is important for the interaction between Hd3a and OsGF14.

FIG. 13 c shows the results of co-expression of various OsFD1 mutants with Hd3a. In the case where the SAP motif was deleted in OsFD1 (“1-191” in FIG. 13 c: a fragment formed of residues 1-191, in which the subsequent amino acids were deleted) or in the case of a serine-to-alanine substitution in the SAP motif (S192A), the amount of OsMADS15 RNA accumulation was found to remarkably lower. In the case of a substitution of serine in the SAP motif to glutamic acid as a phospho-mimicking mutation (S192E), the amount of OsMADS15 RNA accumulation was not found to lower. Those facts suggest that the recognition of phosphorylated serine in the SAP motif in OsFD1 by the 14-3-3 protein is important for the formation of a florigen activation complex.

Example 13-1 Confirmation of Flowering Time in Transgenic Rice

In the same manner as in Example 10, transgenic rice was generated according to Table 3 and its flowering time was confirmed.

A plant was grown at a humidity of 70% in a chamber under short-day (SD) conditions (period including a light period at 30° C. for 10 hours and a dark period at 25° C. for 14 hours) or in a chamber under long-day (LD) conditions (period including a light period for 14 hours and a dark period for 10 hours). In the light period, photoirradiation was performed using a white-light fluorescent tube (400 to 700 nm, 100 μmol·m⁻²·s⁻¹). Rice suspension-cultured cells were maintained as described in Kyozuka and Shimamoto, 1991. A plant was grown at a humidity of 80% in a chamber under short-day (SD) conditions. The number of days to flowering was measured as the number of days to heading (heading stage) of a transgenic plant of T_(o) generation after transferred to SD conditions.

FIG. 14 and Table 3 show the results. An Hd3a-GFP protein derived from transgenic rice having introduced therein prolC::Hd3a::GFP moved from the shoot apex and strongly promoted flowering in the transgenic rice. prolC::Hd3a(R64G)::GFP (gene in which a promoter region in rolC, a coding region in R64G-mutant Hd3a, and a coding region in GFP were conjugated to each other) and prolC::Hd3a(R64G/R132A)::GFP (gene in which a promoter region in rolC, a coding region in R64G/R132A double-mutant Hd3a, and a coding region in GFP are conjugated to each other) were introduced into rice to confirm flowering time. As compared to transgenic rice having introduced therein prolC::Hd3a::GFP (19.6±12.3 days), transgenic rice having introduced therein prolC::Hd3a(R64G)::GFP (44.2±7.8 days) and transgenic rice having introduced therein prolC::Hd3a(R64G/R132A)::GFP (58.8±9.3 days) showed remarkably decreased promotion of flowering, and the latter was almost indistinguishable from a wild-type plant. Those facts suggest that an ability to promote flowering was lost by reduced affinity between Hd3a and 14-3-3.

Next, rice having introduced therein OsFD1 RNAi, transgenic rice expressing OsFD1, and transgenic rice having introduced therein mutant OsFD1 were analyzed. Rice having introduced therein OsFD1 RNAi flowered later than a wild-type. Transgenic rice expressing OsFD1 (pUbq::OsFD1) and transgenic rice having introduced therein mutant OsFD1 S192A (pUbq::OsFD1 (S192A)) were not affected in terms of flowering, and transgenic rice having introduced therein mutant OsFD1 S192E (pUbq::OsFD1 (S192E) (S192 phospho-mimicking)) promoted flowering as compared to a wild-type.

TABLE 3 Days to flowering) Genotype (days) n Wild-type 54.9 ± 9.7 8 prolC::Hd3a::GFP  19.6 ± 12.3 13  prolC::Hd3a(R64G)::GFP 44.2 ± 7.8 6 prolC::Hd3a(R64G/R132A)::GFP 58.8 ± 9.3 21  Wild-type* (wild-type)  69.5 ± 6.0*  4* OsFD1 RNAi*  74.6 ± 4.8*  7* pUbq::OsFD1 58.0 ± 6.1 3 pUbq::OsFD1(S192A) 61.7 ± 2.9 3 pUbq::OsFD1(S192E) 38.3 ± 4.6 8 GF14b GF14e double RNAi 60.1 ± 7.1 14  pUbq::GF14b  55.4 ± 10.9 5 pUbq::NLS::GF14b 51.0 ± 8.9 13  pUbq::GF14e  61.2 ± 11.0 6 pUbq::NLS::GF14e 47.7 ± 8.3 7

Example 13-2 Confirmation of Flowering Time in Transgenic Rice

Transgenic rice was generated in the same manner as in Example 13-1, and its flowering time was confirmed.

A plant (transgenic rice) was grown by the same technique as in Example 13-1, and the number of days to flowering was measured as the number of days to heading (heading stage) of a transgenic plant of T₀ generation after transferred to SD conditions.

FIG. 16 and Table 4 show the results. In the same manner as in Example 13-1, in prolC::Hd3a::GFP transgenic rice, the Hd3a-GFP protein moved from the shoot apex and strongly promote flowering in the transgenic rice. As compared to transgenic rice having introduced therein prolC::Hd3a::GFP, the promotion of flowering was not found in transgenic rice having introduced therein prolC::Hd3a(R64G/R132K)::GFP and transgenic rice having introduced therein prolC:Hd3a(F103A)::GFP (gene in which a promoter region in rolC, a coding region in F103A-mutant Hd3a, and a coding region in GFP were conjugated to each other).

TABLE 4 Days to Genotype Flowering n P WT 55.7 ± 9.4 9 prolC::Hd3a::GFP   19.6 ± 12.3** 13 0.00000018 prolC::Hd3a(R64G)::GFP  44.2 ± 7.8* 6 0.024 prolC::Hd3a(R64G/R132A)::GFP 58.8 ± 9.3 21 0.42 prolC::Hd3a(R64K/R132K)::GFP 61.9 ± 9.7 15 0.13 prolC::Hd3a(F103A)::GFP 54.7 ± 7.9 7 0.83 *P < 0.05, **P < 0.01

INDUSTRIAL APPLICABILITY

The crystal of the present invention provides conformational information on the florigen activation complex important for the elucidation of a mechanism for controlling flowering of a plant, and may be used as a material for additional research on a mechanism for regulating flowering. Further, the flowering of a plant can be artificially and efficiently regulated based on the resultant conformational information, in particular, information on a binding site in the florigen activation complex. Based on the conformational information obtained in the present invention, a transgenic plant, which may be widely utilized for an increase in yield of an agricultural product, an improvement in efficiency of breeding, and the like, can be obtained. Further, a substance that regulates the flowering of a plant can be screened through the utilization of the conformational information on the present invention. Such substance can regulate the growth of a plant under various environments, and hence is considered to be beneficial from the agricultural viewpoint.

(Sequence List)

Seq List¥YGP11-1003PCT_ST25.txt

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130019345A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

1. A method of regulating flowering of a plant, the method comprising at least one of promoting and suppressing formation of a florigen activation complex comprising a complex of a florigen, a 14-3-3 protein, and a bZIP transcription factor by affecting at least one of a binding site between the florigen and the 14-3-3 protein and a binding site between the 14-3-3 protein and the bZIP transcription factor in the florigen activation complex, wherein: the binding site between the florigen and the 14-3-3 protein comprises at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and the binding site between the 14-3-3 protein and the bZIP transcription factor comprises at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO:
 3. 2. A method of regulating flowering of a plant according to claim 1, wherein at least one of the promoting and suppressing of the formation of the florigen activation complex comprises generating a transformant comprising at least one of the following proteins (A) to (C): (A) a florigen having a mutation in at least one binding site between the florigen and a 14-3-3 protein; (B) a 14-3-3 protein having at least one of mutation in at least one binding site between a florigen and the 14-3-3 protein and at least one binding site between the 14-3-3 protein and a bZIP transcription factor; and (C) a bZIP transcription factor having a mutation in at least one binding site between a 14-3-3 protein and the bZIP transcription factor.
 3. A transformant, comprising at least one of the following proteins (A) to (C): (A) a florigen having a mutation in at least one binding site between the florigen and a 14-3-3 protein; (B) a 14-3-3 protein having at least one of mutation in at least one binding site between a florigen and the 14-3-3 protein and at least one binding site between the 14-3-3 protein and a bZIP transcription factor; and (C) a bZIP transcription factor having a mutation in at least one binding site between a 14-3-3 protein and the bZIP transcription factor, wherein: the binding site between the florigen and the 14-3-3 protein comprises at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and the binding site between the 14-3-3 protein and the bZIP transcription factor comprises at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO:
 3. 4. A polynucleotide, which encodes at least one of the following proteins (A) to (C): (A) a florigen having a mutation in at least one binding site between the florigen and a 14-3-3 protein; (B) a 14-3-3 protein having at least one of mutation in at least one binding site between a florigen and the 14-3-3 protein and at least one binding site between the 14-3-3 protein and a bZIP transcription factor; and (C) a bZIP transcription factor having a mutation in at least one binding site between a 14-3-3 protein and the bZIP transcription factor, wherein: the binding site between the florigen and the 14-3-3 protein comprises at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and the binding site between the 14-3-3 protein and the bZIP transcription factor comprises at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO:
 3. 5. A recombinant vector, comprising at least one of the polynucleotides of claim
 4. 6. A method of screening a substance that regulates flowering of a plant, the method comprising any one of the following steps: (1) a step including bringing a candidate substance into contact with any one of a florigen and a 14-3-3 protein, and bringing the candidate substance into contact with any one of the 14-3-3 protein and the florigen, respectively; and (2) a step including bringing a candidate substance into contact with any one of a 14-3-3 protein to which a florigen is bound or unbound and a bZIP transcription factor, and bringing the candidate substance into contact with any one of the bZIP transcription factor and the 14-3-3 protein to which a florigen is bound or unbound, respectively.
 7. A method of screening a substance that regulates flowering of a plant according to claim 6, the method further comprising the following step of: selecting a candidate substance that bring at least one of promoting and inhibiting on binding in at least one of a binding site between the florigen and the 14-3-3 protein and a binding site between the 14-3-3 protein and the bZIP transcription factor in a presence of the candidate substance, wherein: the binding site between the florigen and the 14-3-3 protein comprises at least one site selected from the group consisting of sites of the florigen corresponding to D62, M63, R64, P96, T98, F103, and R132 in SEQ ID NO: 1 and sites of the 14-3-3 protein corresponding to F200, D201, I204, E212, Y215, R226, and D227 in SEQ ID NO: 2; and the binding site between the 14-3-3 protein and the bZIP transcription factor comprises at least one site selected from the group consisting of sites of the 14-3-3 protein corresponding to K51, R58, F121, R131, L224, and D227 in SEQ ID NO: 2 and sites of the bZIP transcription factor corresponding to R189 to F195 in SEQ ID NO:
 3. 8. A method of screening a substance that regulates flowering of a plant according to claim 6, wherein: the florigen comprises a florigen polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177, in an amino acid sequence set forth in SEQ ID NO: 1; the 14-3-3 protein comprises a 14-3-3 protein polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256, in an amino acid sequence set forth in SEQ ID NO: 2; and the bZIP transcription factor comprises a bZIP transcription factor polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195, in an amino acid sequence set forth in SEQ ID NO:
 3. 9. A polypeptide fragment, which is selected from the following: (i) a novel florigen polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177, in an amino acid sequence set forth in SEQ ID NO: 1; (ii) a novel 14-3-3 protein polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256, in an amino acid sequence set forth in SEQ ID NO: 2; and (iii) a novel bZIP transcription factor polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195, in OsFD1 set forth in SEQ ID NO:
 3. 10. A polynucleotide, which encodes any one of the polypeptide fragments (i) to (iii) of claim
 9. 11. A florigen activation complex, comprising a florigen polypeptide fragment bound to a bZIP transcription factor polypeptide fragment via a 14-3-3 protein polypeptide fragment, wherein the florigen polypeptide fragment, the 14-3-3 protein polypeptide fragment, and the bZIP transcription factor polypeptide fragment comprise the polypeptide fragments (i) to (iii) of claim 9, respectively.
 12. A crystal of a florigen activation complex, comprising a florigen bound to a bZIP transcription factor via a 14-3-3 protein.
 13. A crystal of a florigen activation complex according to claim 12, wherein the crystal has a space group of P1, P6522, or P4 and lattice constants of a=74 to 158 Å, b=64 to 158 Å, c=96 to 500 Å, α=66 to 90°, β=85 to 90°, and γ=75 to 120°.
 14. A method of producing the crystal of a florigen activation complex according to claim 12, the method comprising the steps of: crystallizing a solution containing a complex of a florigen and a 14-3-3 protein by a vapor diffusion method using a precipitant solution containing at least as a precipitant 5 to 35 vol % polyethylene glycol; collecting the resultant crystal; and obtaining a crystal of a florigen activation complex by incubating the resultant crystal using a precipitant solution containing at least as a precipitant 5 to 35 vol % polyethylene glycol and containing a bZIP transcription factor.
 15. A method of screening a substance that regulates flowering of a plant by at least one of designing and selecting a candidate substance having a function of regulating an activity of a florigen activation complex using a computer, the method comprising the steps of: (a) causing storage means to store conformational information obtained from the crystal of a florigen activation complex of claim 12; (b) causing deriving means to derive a three-dimensional conformation model based on the conformational information; (c) causing calculation means to calculate an interatomic distance in the derived three-dimensional conformation model; and (d) causing calculation means to calculate, based on the calculated interatomic distance, conformational information on a candidate substance being capable at least one of enhancing and inhibiting against at least one of binding between a florigen and a 14-3-3 protein and binding between a 14-3-3 protein and a bZIP transcription factor, and to bring at least one of designing and selecting the candidate substance.
 16. A method of screening a substance that regulates flowering of a plant according to claim 7, wherein: the florigen comprises a florigen polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 62 to 132, starts with one of amino acids at positions 1 to 6, and ends with one of amino acids at positions 165 to 177, in an amino acid sequence set forth in SEQ ID NO: 1; the 14-3-3 protein comprises a 14-3-3 protein polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 51 to 227, starts with one of amino acids at positions 1 to 5, and ends with one of amino acids at positions 230 to 256, in an amino acid sequence set forth in SEQ ID NO: 2; and the bZIP transcription factor comprises a bZIP transcription factor polypeptide fragment comprising an amino acid sequence which comprises at least a sequence of amino acids at positions 189 to 195, starts with one of amino acids at positions 182 to 188, and ends with the amino acid at position 195, in an amino acid sequence set forth in SEQ ID NO:
 3. 17. A method of producing the crystal of a florigen activation complex according to claim 13, the method comprising the steps of: crystallizing a solution containing a complex of a florigen and a 14-3-3 protein by a vapor diffusion method using a precipitant solution containing at least as a precipitant 5 to 35 vol % polyethylene glycol; collecting the resultant crystal; and obtaining a crystal of a florigen activation complex by incubating the resultant crystal using a precipitant solution containing at least as a precipitant 5 to 35 vol % polyethylene glycol and containing a bZIP transcription factor.
 18. A method of screening a substance that regulates flowering of a plant by at least one of designing and selecting a candidate substance having a function of regulating an activity of a florigen activation complex using a computer, the method comprising the steps of: (a) causing storage means to store conformational information obtained from the crystal of a florigen activation complex of claim 13; (b) causing deriving means to derive a three-dimensional conformation model based on the conformational information; (c) causing calculation means to calculate an interatomic distance in the derived three-dimensional conformation model; and (d) causing calculation means to calculate, based on the calculated interatomic distance, conformational information on a candidate substance being capable at least one of enhancing and inhibiting against at least one of binding between a florigen and a 14-3-3 protein and binding between a 14-3-3 protein and a bZIP transcription factor, and to bring at least one of designing and selecting the candidate substance. 